THE JOURNAL OF BIOLOGICAL, CHEMISTRY Vol. 249, No. 12, Issue of June 25, PP. 3737-3745, 1974

Printed in U.S.A.

pH Jump Studies of Glutamate Decarboxylase

EVIDESCE FOR A pH-DEPENDENT CONFORR’IATION CHANGE*

(Received for publication, November 26, 1973)

MARION H. O’LEARYS AND WALTER BRUMMUND, JR.$

From the Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706

SUMMARY

The catalytically active form of bacterial glutamate de- carboxylase has an absorption maximum at 420 nm due to the presence of the enzyme-pyridoxal 5’-phosphate Schiff base. Above pH 5.5 the absorption maximum is at 340 nm, and the enzyme is inactive. If the pH of a solution of the enzyme is rapidly changed from 6.5 to 4.0 in a stopped flow apparatus, the change in absorption spectrum occurs over a period of several seconds. The change is general acid cata- lyzed and occurs in two steps. The first is a rapid conforma- tion change of the enzyme induced by the addition of 3 pro- tons to the enzyme. The second is general acid catalyzed formation of the catalytically active form of the enzyme.

If the pH of a solution of the enzyme is rapidly increased from 4.0 to 7.0, the rate of the change in absorption spectrum can also be measured. The change is not buffer catalyzed, but its rate increases rapidly with increasing pH. The mechanism of this change involves an initial rate-determining conformation change of the enzyme induced by dissociation of 3 protons, followed by rapid formation of the equilibrium high pH form of the enzyme. The rate of the conformation change can also be measured on enzyme which has been re- duced by sodium cyanoborohydride.

From the kinetics of the pH jump reactions and other fac- tors we conclude that the high pH form and the low pH form of glutamate decarboxylase differ by a protein conformation change and by a change in the covalent structure of the bound coenzyme. At low pH the coenzyme is bound in the usual ketoenamine form. At high pH a sulfhydryl group of the enzyme adds to the aldehyde carbon forming an aldamine.

Many anions affect the properties of glutamate decar- boxylase. Chloride ion increases the rate of transformation of the high pH form of the enzyme into the low pH form and decreases the rate of transformation of the low pH form into the high pH form. These effects occur because anions are

* This study was supported by Grant NS-07657 from the Na- tional Institute of Neurological Diseases and Stroke and by a grant from the University of Wisconsin Graduate School. The enzyme preparation was made possible by National Institutes of Health Grant FR00226.

f To whom correspondence should be addressed. Fellow of the Alfred P. Sloan Foundation, 1972 to 1974.

0 From the Ph.D. Thesis of W. Brummund, Jr., University of Wisconsin, 1973. Complete kinetic data will be found in this thesis, which is available from University Microfilms, Ann Arbor, Michigan.

able to bind to the low pH conformation of the enzyme but not to the high pH conformation.

The absorption spectrum of the pyridoxal 5’-phosphate-de- pendent bacterial glutamate decarboxylase (EC 4.1.1.15) de- pends on the pH (1). At low pH the enzyme absorbs at 420 nm, is catalytically active, and can be reduced by means of NaBHI. Above pH 6 the enzyme absorbs at 340 nm, is catalytically inac- tive, and cannot be reduced with NaBH4 (2, 3).

The absorption spectrum of the enzyme changes abruptly with pH. A plot of absorbance at 420 nm or 340 nm versus pH gives a titration curve which is much steeper than an ordinary titration

curve and which most readily fits a simultaneous 4-proton tran- sition

E340 + 4H+ + H4E.420

whea F&o is the high pH form of the enzyme (absorption maxi- mum 340 nm) and H4EhZ0 is the low pH form of the enzyme (absorption maximum 420 nm). The midpoint of the transition is at pH 5.61 in acetate or phosphate buffer (1) and at 5.30 in pyridinium sulfate buffer (4).

The low pH chromophore of glutamate decarboxylase is a Schiff base between pyridoxal-P and an t-amino group of a ly- sine residue of the enzyme (2). Numerous model studies have demonstrated that the Schiff base exists principally as a ketoen-

amine (Scheme I), rather than a hydrogen-bonded imine (4, 5). The structure of the high pH chromophore is less certain. An absorbance at 340 nm is unusual, altiough not unprecedented, for pyridoxal-P bound to proteins. We (6) and others (2) have argued that the 340 nm absorbance is due to the presence of an aldaminel (Scheme 1) formed by the addition of some group -X to the 4’ carbon atom of the low pH form. The identity of the hypothetical group -X remainsa subject of specu- lation. Model aldamines formed by addition of various groups to imines are known (6).

In addition to the differences in covalent structure, the low pH form and the high pH form of glutamate decarboxylase may also

1 The nomenclature of these compounds is particularly unfortu- nate. The compound formed by reaction of an aldehyde with a primary amine is called an aldimine, or occasionally a Schiff base. The compound formed by addition of some group to the aldimine (as in Scheme 1) is called an aldamine.

3737

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I HC

/NH

=03POCH2

low pH high pH

SCHEME 1. The nature of the bound coenzyme in glutamate decarboxylase.

differ in conformation. The abruptness of the pa-dependent

spectral change suggests that a pH-dependent conformational change occurs. Optical activity data also suggest the occurrence of such change. The 420 nm absorption of the low pH form of the enzyme has a strong positive Cotton effect, but the 340 nm absorption of the high pH form of the enzyme has only a very weak Cotton effect (7, 8). When glutamate decarboxylase is reacted with hydroxylamine, the product is an oxime of pyri- doxal-P bound to the enzyme. The 380 nm absorption of this material shows a change in sign of its circular dichroism with pH, even though its ultraviolet spectrum is unaffected (7).

A number of properties of glutamate decarboxylase are af- fected by anions. The rate of enzymatic decarboxylation (1, 4, 9) and the rate of coenzyme binding to the apoenzyme (10) are affected by chloride ion. Anions also affect the ultraviolet spectrum (7, 8) and CD spectrum (7) of the enzyme. The mid- point of the pH-dependent spectral transition is affected by anions (1, 4). NMR studies of anion binding to the enzyme in- dicate that bromide and chloride bind at a site distinct from the active site (9).

In this paper we report a study of the differences between the high pH and low pH forms of glutamate decarboxylase by a pH jump technique. The pH jump experiment involves the meas- urement of the rate of change of the enzyme spectrum following a rapid change in the pH of the enzyme. This rapid change is achieved by mixing the enzyme in dilute buffer with concen- trated buffer of a different pH. The dependence of the pH jump transition rate on various parameters provides direct evidence for the postulated differences between the high and low pH forms of glutamate decarboxylase. Salt effects on the pH jump rate provide additional information about the pH jump mechanism and about the effect of anions on the enzyme.

EXPERIMENTAL PROCEDURE

Materials-L-Glutamic acid, pyridoxal-P, and Trizma (2.amino- 2.hydroxymethyl-1,3-propanediol) base were obtained from Sigma Chemical Co. 2,2’-Dithiodipyridine and dithiothreitol were from Aldrich Chemical Co. Sodium cyanoborohydride was obtained from Alfa Inorganics, Inc. Other materials were reagent grade. Water was purified with a Millipore Super Q water purification system or deionized and then distilled twice.

Glutamate decarboxylase was isolated from locally grown Escherichia coli (ATCC 11246) by our published procedure (11). Before use, enzyme samples were heated for 15 min at 50”, centri- fuged, and desalted on Sephadex G-25.

Melhods-Ultraviolet spectra were measured with a Cary 15 recording spectrophotometer. Kinetics of slow reactions and routine absorbance were measured with a Gilford model 222 spec- trophotometer attached to a Beckman DU monochromator and a Sargent model SRG strip chart recorder. Fluorescence spectra were measured on an Aminco-Bowman SPF-2 spectrophoto- fluorometer attached to a Hewlett-Packard 7035B recorder at 20”. All pH measurements were made with a Radiometer PHM 26 pH

meter by the two-buffer method. All buffers contained 10-h M dithiothreitol.

Glutamate decarboxylase was assayed with a Gilson differential respirometer at 37” (11). Protein concentrations were obtained from absorbance readings by means of the correction factor ab- sorbance 1.0 = 1.7 mg per ml (12).

Sulfhydryl titrations were performed with enzyme at approxi- mately 1 mg per ml and 2,2’-dipyridyldisulfide -2 mM. Acetate buffer. 0.1 M. DH 4.5. and nhosnhate buffer. 0.1 M. DH used, and the reaction was moni’tored at 343 hm (13).L

6.5. ’

were

Reduced Glutamate Decarboxylase-Ten milligrams of enzyme were dissolved in 3.5 ml of 0.05 M acetate buffer, pH 4.8, contain- ing 10e4 M dithiothreitol and 5 X lo-& M pyridoxal-P. Then 0.5 ml of 0.14 M NaBH&N was added in 0.1.ml portions over a period of 1 hour at 20”. After gel filtration the residual activity of the enzyme was about 37, that of the native enzyme.

The pK, of the reduced enzyme was measured by successive measurements of the fluorescence spectrum of a 3-ml portion of the enzyme (0.25 mg per ml) after small pH increments.

pH Jump Experiments-All pH jump rate measurements were made at 2010” with a Durrum-Gibson stopped flow spectrophotom- eter eauinned with a Kel-F block with zero disnlacement valves. a high-intensity xenon lamp and a tungsten lamp, and a movable photomultiplier which can be used for both fluorescence and ab- sorbance measurements. The output from the photomultiplier and amplifier was recorded with a Tektronix 546B storage oscillo- scope and Polaroid camera. All kinetic measurements on the native enzyme were made in the absorbance mode. Rates were calculated from the absorbance change at 420 nm, except where otherwise noted. Kinetic measurements on the reduced enzyme were made in the fluorescence (and $&Z’) mode with the use of 280 nm light from the xenon lamp for excitation and a Corning CS O-52 filter (50$& transmittance at 360 nm) between the cell and the photomultiplier.

Photographs of oscilloscope traces were converted to digital form by means of a D-Mac model PF-1OB computer digitizer (Ed- win Industries) which punched the data onto computer cards. These data were then computer plotted by means of the usual first order rate equation. Best fit rate constants were determined from these plots by eye.

In the pH jump experiments, a weakly buffered enzyme solution at one pH is mixed with an equal volume of a concentrated buffer solution at another pH. The weakly buffered solution always contained 0.01 M buffer, lo+ M dithiothreitol, and 10e6 M pyri- doxal-P. The concentrated buffer was 0.1 to 0.4 M and contained lo-‘M dithiothreitol. All buffers were degassed prior to use. The final pH of the solutions after the pH jump was determined by mixing equal quantities of the two buffers.

RESULTS

pH Jump Down-When the pH of a solution of glutamate de- carboxylase is rapidly changed from 7.0 to 4.0 by mixing enzyme in dilute pH 7 buffer with concentrated pH 4 buffer in a stopped flow machine, an increase in the absorbance of the solution at 420 nm and a corresponding decrease at 340 nm can be observed over a period of several seconds. Under most conditions these changes follow first order kinetics. The rate constant calculated from the absorbance change is independent of the wavelength monitored, independent of the initial pH of the enzyme solution over the range from 6.0 to 7.1, and independent of the enzyme

concentration over the range from 0.01 mg per ml to 1.9 mg per ml. The presence of low4 M dithiothreitol and 10e5 M pyridoxal-P improves the quality of the results, but otherwise has no effect on the rat,e, so these two substances were included in most meas- urements.

The calculated rate constant is independent of initial pH and enzyme concentration, but increases with decreasing final pH. At constant pH, the rate increases with increasing buffer con- centration. Kinetic data spanning a range of buffer concentra- tions and final pH values in sodium acetate buffers are given in Fig. 1.

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0.8

0.6 -7 VI

2 2

0.4

0.2

C

FINAL pH

FIG. 1. First order rate constants for the pH jump down in acetate buffer at 20.0”. Glutamate decarboxylase in 0.01 M

sodium acetate buffer, pH 6.0, was mixed 1:l with varying con- centrations of sodium acetate buffer of lower pH. After mixing, the enzyme concentration was 0.58 mg per ml, and the total buffer concentrations (acetic acid plus acetate) were 0.055 M (O), 0.105 M (O), 0.155 M (A), and 0.205 M (A). The curves are theoretical curves for the four buffer concentrations calculated from Table I and Equation 4.

A number of possible mechanisms for the pH jump transition was tested for fit to the kinetic data. One-step mechanisms of the type

E340 + H+ ___j HE420 (2)

in which E340 is the high pH form of the enzyme and HEdZO is the low pH form of t.he enzyme, did not fit the pH dependence of the observed rate. Particularly poor fit was obtained between pH 4.6 and 5.2. Inclusion of terms for buffer catalysis did not result in a satisfactory fit.

Equation 2 does not account for the known 4-proton difference between the two forms of the enzyme (Equation 1). When Equation 2 was modified to include 2, 3, or 4 protons, the fit was even worse than with only 1 proton.

The simplest mechanism which fits the kinetic data satisfac- torily is the two-step mechanism

+-!L k E340 + 3H - 83E340 - “qE420 (3)

in which the first step is a rapid equilibrium and the second step is rate determining. A fourth proton is added in the second step, which is buffer catalyzed. HJZzaO is a metastable form of the enzyme which has the same spectrum as EsdO, but differs from it in conformation and proton content (vide infra).

The data in Fig. 1 show clearly that the reaction is buffer cata- lyzed, so the observed rate of the pH jump reaction (k& is given by

TABLE I

Best jit values of rate constants for pH jump down

Rate constants were obtained from least squares fitting of ex- perimental rate constants to Equation 4. Values of K in acetate buffers were obtained by varying the value of K used as part of the fitting procedure until an optimum fit was obtained. Values of K in formate buffers were assumed to be equal to those in ace- tate buffers. No evidence was obtained for base catalysis in any case (kOH = kB = 0).

Buffer Chloride concen- tration

PL ko kn kA Correlation coefficient

Acetate. ...... Acetate. ..... Formate. .... Formate ......

M s-1 M-1 s-1

0 15.2 0 4000 0.1 15.6 0.62 4800 0 15.2 0 4600 0.1 15.2 0.79 5400

3.4 0.994 2.5 0.998 41 0.982 13 0.997

where HA is acetic acid, and A- is acetate ion. This equation includes the possibility of both general acid and general base catalysis. The first step in Equation 3 is assumed to be rapid.

After a preliminary fitting by hand, the kinetic data in Fig. 1 were computer fitted to Equation 4 by means of a standard multi- ple linear least squares program. The computer was given a set of data for each point on Fig. 1 including the observed rate COIN-

stant and the concentrations of acetic acid, acetate ion, hydrogen ion, and hydroxide ion. In addition, a value of K was assumed for the entire set of data. The computer calculated best fit values of ko, kH, kOH, k,, and kg.

Because of the nature of the fitting procedure, it was not possible to have the computer optimize the value of K; instead, this was done externally by giving the computer a series of values of K and finding the value for which the best over-all fit was ob- tained. In the present set of data, the value of pK is probably accurate to about hO.4.

The choice of 3 protons in the first step of Equation 3 was dictated by two factors. In the first place, the addition of 3 protons in the first step and a fourth proton in the second makes this mechanism consistent with the known 4-proton difference between the two equilibrium forms of the enzyme (Equation 1). In addition, different numbers of protons were tried as part of the fitting procedure discussed above, and an optimum fit was obtained for 3 protons.

The rate constants derived from this fitting procedure are given in Table I. In all cases, kOH and lig were zero. That is, the pH jump reaction is subject to general acid catalysis, but not to gen- eral base catalysis or specific base catalysis. In addition, k. was zero in this system, although this was not so in some later situations. The curves given in Fig. 1 are theoretical curves de- rived from the rate constants given in Table I.

Spectrum of H3ES4,,-The spectrum of intermediate H3ESd0 was obtained by extrapolation. The pH jump to a final pH of 4.0 was repeated a number of times and absorbance versus time was recorded at various wavelengths from 300 nm to 450 nm. The absorbances were extrapolated to the time of mixing, and these extrapolated values were used to construct the spectrum of the intermediate H3ESd0, which should be present at high concentra- tion right after mixing. The spectrum so obtained has an ab- sorption maximum at 340 nm and is not significantly different from that of E340.

1 k obs =

1 + K/[H+13 (k. + kH tH+l + koH [OH-I + LAtHAl + k@-I)

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0

FINAL pH

FIG. 2. First order rate constants for the pH jump down in formate buffer at 20.0”. Glutamate decarboxylase in 0.01 M

sodium formate buffer, pH 6.0, was mixed 1:l with varying con- centrations of sodium formate buffer. After mixing, the enzyme concentration was 0.32 mg per ml, and the total buffer concentra- tions were 0.055 M (O), 0.105 M (O), O.i55 M (A), and 0.205 M (A). The curves are theoretical curves for the four buffer concentra- tions calculated from Table I and Equation 4.

pH Jump Down in Formate Bugler-The pH jump studies were repeated in sodium formate buffers at various concentrations and pH values. The rates in formate buffers are generally somewhat higher than those in the corresponding acetate buffers at the same pH.

When the final pH was 4.5 or greater, the change in absorbance with time followed excellent first order kinetics. However, be- low pH 4.5, the first order plots were slightly curved, as if two different kinetic processes were going on simultaneously. This phenomenon became more pronounced as the over-all rate be- came faster. The curvature was independent of enzyme con- centration, independent of the presence or absence of added pyridoxal-P, independent of the wavelength monitored, and independent of the particular enzyme preparation used.2 After an initial curved portion, the first order rate plot usually became quite straight, and this straight portion was used to calculate the rate constant.

The pH dependence and buffer dependence of the pH jump rates in formate buffers were analyzed in precisely the same way as those in acetate buffer. The kinetic data are shown in Fig. 2,

2 There are several possible explanations for this phenomenon, none of which can convincingly be eliminated. The faster process might be due to the presence of a small amount of a different form of the enzyme which undergoes the pH jump more rapidly than the major form. Alternatively, the pH jump reaction might involve cooperative interaction of more than one subunit of the enzyme. The third (and perhaps most likely) alternative is that when the rate of the second step in Equation 3 becomes sufficiently fast, the rate of the first step is no longer fast enough to maintain it at equilibrium. A quantitative analysis of this mechanism is ex- tremely difficult (14). In such a mechanism, the slope of the first order plot after the initial curvature still gives the rate constant kobs (Equation 4).

I I I I I I

4.0 4.4 4.8 5.2

FINAL pH

FIG. 3. First order rate constants for the pH jump down in acetate buffer containing 0.1 M NaCl at 20.0”. Conditions are identical with t,hose of Fig. 1, except that all solutions contained 0.1 M NaCl.

along with theoretical curves derived from the mechanism of Equation 3 and the best fit rate constants given in Table I. Be- cause of the lack of buffering capacity of formic acid near pH 5, fewer data were obtained above pH 4.5 than in acetate buffer; as a result, it was not possible to obtain an accurate value of K (Equation 3) from these kinetic data. Instead, the best fit value from the acetate data was used.

pN Jump Down in Presence of Chloride-The rate of the pH jump down is increased by the presence of chloride ion. As in the case of formate buffer, some of the first order rate plots were slightly curved. The rate constants were obtained from the later, straight portions of these plots. Results of a series of meas- urements of the rate of the pH jump down in acetate buffers con- taining 0.1 M NaCl are given in Fig. 3. The data were assumed to conform to Equation 3 and were analyzed accordingly. The derived rate constants are given in Table I. The equilibrium constant K is somewhat lower in the presence of chloride than in its absence.

In addition to the effect on K, the difference in the rate of the pH jump in the presence and in the absence of chloride is prin- cipally due to the occurrence of an uncatalyzed transition ap- pearing as a term k,, in Equation 4. This term in zero in the absence of chloride, but in the presence of chloride it contributes appreciably to the observed rate. Rate constants kH and k, are altered only slightly by the presence of chloride.

The pH jump to a final pH of 4.0 was studied over a range of chloride concentrations. The results (Fig. 4) can be most easily interpreted if it is assumed that chloride does not bind to E340, but binds to H3ES4,, with a dissociation constant of 8 mM.

The same effect of chloride ion on the rate was obtained whether chloride was present in only one buffer before mixing, or in both buffers. Thus, the chloride-binding equilibrium is established rapidly compared to the time of the pH transition.

The pH jump rate was measured in a series of formate buffers in the presence of 0.1 M NaCl. The data are shown in Fig. 5 and the derived kinetic pa,rameters in Table I. As in the acetate

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0.8

Oi 0 0.05

FINAL NaCl CONCENTRATION, M

FIG. 4. Effect of chloride on the pH jump down. Glutamate decarboxylase in 0.01 M sodium acetate buffer, pH 6.0, was mixed I:1 with sodium acetate buffer. After mixing, t,he enzyme con- centration was 0.55 mg per ml, the buffer concentration was 0.105 M, and the pH was 4.47. In one set of experiments (A) both the initial enzyme solution and the initial buffer contained N&l at t,he indicated concentration. In the other set (0) the initial enzyme

TABLE II

Effects of anions on pH jump The pH jump down was repeated a number of times with ace-

tate buffer at final concentration 0.105 M, pH 4.49, containing 0.1 M concentrations of the sodium salts of various anions. The pH jump up was conducted with phosphate buffer at a final con-

centration of 0.10 M, pH 7.08, containing 0.1 M concentrations of the sodium salts of various anions.

Buffer only

F- soa* BFd-

Cl- NO,- Br-

I- SCN-

bobs

pHjumpdown ~Hjumpup

s-1

0.57 0.16

0.90 1.6 1.1 0.55

1.6 3.7 3.0

9.5 9.2

8.8 1.6 0.78 0.55

0.27 0.14 0.11

buffers, the principal effect of adding chloride is to cause the ap- pearance of a kinetic term ko. It was not possible to determine whether the presence of chloride in the formate buffers affected the value of K.

Effects of Other Anions--Many other anions in addition to chloride affect the rate of the pH jump transition. Rate con- stants for the pH jump to pH 4.5 in 0.05 M acetate buffer in the presence of 0.1 M concentrations of the sodium salts of various anions are shown in Table II.

pZi Jump Up-When the pH of a solution of glutamate decar-

0.5: 4.0 4.4 4.8

FINAL pH

solution contained no NaCl, and the acetate buffer contained twice the indicated concentration of NaCI.

FIG. 5. First order rate constants for the pH jump down in formate buffer containing 0.1 M NaCl at 20.0”. Conditions are identical with those of Fig. 2, except that all solutions contained 0.1 M NaCl.

boxylase is rapidly increased from 4.5 to 7.0, the change in the absorption spectrum of the enzyme occurs over a period of sev- eral seconds. The absorbance change follows first order kinetics. The rate of the transition is independent of the initial pH of the enzyme over the range of initial pH from 4.1 to 4.9 and is inde- pendent of the concentration of the enzyme from 0.05 mg per ml to 1.1 mg per ml. No buffer catalysis is observed, although the rates vary slightly with ionic strength.

The pH dependence of the pH jump rate is shown in Fig. 6. When the final pH is between 5.7 and pH 6.4 the slope of a plot of log rate versus final pH is about three, indicating that 3 protons are being transferred in or prior to the rate-determining step. Above pH 7 the slope is less than 1.

An attempt was made to detect an intermediate in the pH

jump up by extrapolat.ion of absorbances at various wavelengths to the time of mixing, as was done in the pH jump down. The spectrum so obtained is identical with that of HdE420, the low pH form of the enzyme.

The kinetic data for the pH jump up are consistent with the scheme

H4E420 slow, 3H+ + HE420 fast

' E340 + H+ (5)

in which the first step is rate determining. The pK, for the 3-proton transition in the first step is 19.2; thus, the transition is half-complete at pH 6.4.

Eflect of Anions on pH Jump Up-The presence of 0.1 M NaCl in the pH jump up shifts the pH rate profile to higher pH (Fig. 6). The same anions that cause an increase in the rate of the pH jump down cause a decrease in the rate of the pH jump up (Ta- ble II). The rate of the pH jump up approaches zero at high chloride concentrations (Fig. 7), indicating that H4E42,, cannot undergo the pH jump transition when chloride is bound. A

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FINAL pt-t

FIG. 6. First order rate constants for the pH jump up in phos- phate buffer at 20.0”. Glutamate decarboxylase in 0.01 M acetate buffer, pH 4.87, was mixed 1:l with Tris-phosphate buffer (A) or sodium phosphate buffer (0: X). After mixing, the enzyme concentration was 0.44 mg per ml, and the buffer concentration was 0.1 M. The rates on the higher pH curve were measured in the presence of 0.1 M NaCl (X, A).

FIG. 7. Effect of chloride on the pH jump up. Glutamate de-

FINAL NaCl CONCENTRATION, M

carboxylase in 0.01 M sodium acetate buffer, pH 4.9, was mixed 1:l with sodium phosphate buffer. After mixing, the enzyme concentration was 0.31 mg per ml, the pH was 7.08, and the buffer concentrat.ion was 0.1 M. In one set of experiments (A) both the initial enzyme solution and the initial buffer contained NaCl at the indicated concentration. In the other set (0) the initial enzyme solution contained no NaCl, and the phosphate buffer con- tained twice the indicated concentration of NaCl.

/ /’

.’

6.0 7.0

FINAL pH

8.0

FIG. 8. First order rate constants for the pH jump up with reduced glutamate decarboxylase. Reduced glutamate decar- boxylase in 0.01 M acetate buffer, pH 4.8, was mixed 1:l with Tris-phosphate buffer. After mixing, the enzyme concentration was 0.50 mg per ml and t,he buffer concentration was 0.1 M. - - -, pH jump reaction of the unreduced enzyme under the same condi- tions.

dissociation constant of 17 mM for the binding of chloride to

H~EQ~ was calculated from the data in Fig. 7.

pH Jump Studies of Reduced Glutamate Decarbox$ase-Gluta- mate decarboxylase was reduced with NaBH&N. The reduced enzyme, which has a secondary amino group connecting pyridox- amine 5’-phosphate to the protein (2), has a pH-independent absorption maximum at 330 nm. A fluorescence emission at 390 nm from the bound, reduced coenzyme is observed if the en- zvme is excited at 280 nm or 330 nm. When 280 nm excitation

PH

FIG. 9. pH titration of reduced glutamate decarboxylase. Measurements were made by fluorescence with 280 nm excitation and 390 nm emission. The curve is a theoretical curve for simul- taneous dissociation of 4 protons (Equation 1). Fluorescence is given in arbitrary units.

is used, the intensity of the 390 nm emission varies by about a factor of two between pH 4 and pH 7.

When reduced enzyme at pH 6 was mixed with concentrated buffer at pH 4 no transition could be observed in the time range of the stopped flow machine. However, when reduced enzyme at pH 4.5 was mixed with high pH buffer, a fluorescence change could be observed which followed first order kinetics. The rate of this transition was very similar to the rate of the pH jump transition on the native enzyme (Fig. 8). The presence of 0.1 M NaCl has the same inhibiting effect on the rate of the pH jump with the reduced enzyme as with the native enzyme.

Titration of Reduced Enzyme-The fluorescence spectrum of reduced glutamate decarboxylase was recorded as a function of pH over the range pH 5.5 to 7.0. The titration curve (Fig. 9) shows the same characteristic abrupt change as does that of the native enzyme (1, 4). The midpoint of the titration is at pH 6.2 in the presence of 0.1 M NaCl.

Suljhydryl Determination-Glutamate decarboxylase was ti-

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trated at pH 6.5 and at pH 4.5 with 2,2’-dithiodipyridine (13), a sulfhydryl reagent that can be used over a range of pH values. Only one sulfhydryl group could be titrated per 50,000 daltons at either pH. The catalytic activity and absorption spectrum were unchanged following the modification.

DISCUSSION

Our studies require that the two forms of glutamate decar- boxylase differ both by a conformation change and by a change in covalent structure. The evidence for these two points is discussed in the first section, along with a general description of the nature of the differences between the two states of the en- zyme. In the following sections the mechanisms of the pH jump down and the pH jump up are discussed.

Two Forms of Glutamate Decarboxylase-This work provides clear evidence that the pa-dependent change in the spectrum of glutamate decarboxylase is in part the result of a change in conformation of the enzyme. The change in the fluorescence spectrum of the reduced enzyme with pH must be due to a con- formation change because no change in covalent structure is possible in this case. The spectral changes are not due to ioniza- tions. The absorption spectrum does not change in the pH range under consideration, and the change in the fluorescence spectrum does not correspond to any pa-dependent change which has been observed in the fluorescence of pyridoxamine 5’-phos- phate in solution (15). The fluorescence being observed (excita- tion at 280 nm, emission at 390 nm) is probably due to light absorption by tyrosine or tryptophan residues of the enzyme, nonradiative energy transfer to the reduced coenzyme, and emis- sion from the coenzyme. The efficiency of energy transfer is sensitive to the separation and relative orientations of the two chromophores (16), and so might reasonably be changed by a conformation change.

Kinetic evidence for the conformation change also exists. The slow transition of the reduced enzyme from a low pH form to a high pH form must result from a conformation change, be- cause there is no structural difference (with the exception of protonations) between the two forms.

The pH-dependent conformation change in glutamate decar- boxylase is probably the cause of the abrupt change in the spec- trum of the enzyme with pH. It is striking that both the re- duced enzyme and the native enzyme undergo this same change. Thus, although aldamine formation and decomposition are associated with the conformation change in t,he unreduced en- zyme, the conformation change occurs even when aldamine formation cannot occur.

The equilibrium constant for the conformation change of glutamate decarboxylase can be obtained without the complica- tions of chemical changes which occur in the native enzyme from the spectrophotometric titration of the reduced enzyme. This change is half-complete3 at pH 6.2. This number is similar to the 6.4 observed in the pH jump up on the native enzyme, but it is quite different from the 5.1 observed in the pH jump down on the native enzyme, probably because the aldamine works to prevent the conformation change.

In addition to the difference in conformation between the high pH and low pH forms of glutamate decarboxylase, there must also be a difference in covalent structure between the two forms. General acid catalysis of the conversion of Es40 to H$.+20 was observed for two different acids over a range of acid con-

s This number was obtained in the presence of 0.1 M NaCl. By analogy with the results obtained on the native enzyme, this number would probably be about 6.0 in the absence of chloride.

centrations. The rate of the pH jump down does not depend on the concentration of acetate ion or formate ion (even though a number of other monovalent anions have a striking effect on the rate), but it does depend on the concentrations of the con- jugate acids. Hydronium ion, acetic acid, and formic acid con- form to the Bronsted catalysis law, as expected. General acid catalysis of the pH jump down is only possible if the high pH form of the enzyme is an aldamine (Scheme 1) formed by the addition of some enzyme group -X across the carbon-nitrogen double bond of the Schiff base. Decomposition of such struc- tures has been shown to be general acid catalyzed in a number of model systems (17), but the present study represents the first such catalysis of an enzyme structure change.

What is this group -X which adds across the carbon-nitrogen double bond to form the aldamine? The possibilities include water, an amino group of a lysine, a ring carbon of histidine, the -OH of a serine or threonine, and the -SH of a cysteine. Only the last of these possibilities is consistent with all available data, as we show in the following discussion.

The possibility that -X is -OH is remote. Such a structure is presumably formed as an intermediate in the reaction of apo- glutamate decarboxylase with pyridoxal-P, and similar struc- tures are presumed to be intermediates in reactions of pyridoxal-P and other aldehydes with primary amines (18, 19), but there is no precedent for the occurrence of such a structure as a stable species.

We previously argued that the aldamine was probably formed by addition of a second lysine-NH2 to the carbon-nitrogen double bond of the Schiff base (6), although we noted at that bime that there was an entropy problem with such a structure. The oc- currence of general acid catalysis in the pH jump down eliminates this possibi1it.y. Studies of gem-diamines have shown that the higher of the two pK, values of the diamine is similar to that of a corresponding simple amine (20); thus, if -X were -NHR of a second lysine residue, one of the nitrogen atoms of the alda- mine should be protonated at pH 3 to 5, and general acid-cata- lyzed decomposition of the aldamine would not occur.

Although histidine reacts with pyridoxal-P to form an al- damine-type structure (21, 22), this reaction is probably not reversible.

Addition of a serine or threonine -OH to a Schiff base to form an aldamine is possible, but such adducts have never been ob- served. Reaction of pyridoxal-P with serine or threonine results only in the formation of a Schiff base (22, 23).

Thus, the most likely candidate for the aldamine-forming group is the -SH of a cysteine residue. Such compounds are readily formed in the reaction of pyridoxal-P with cysteine (23, 24) and other aminomercaptans (25). Glutamate decarboxyl- ase has 10 cysteine residues per subunit (mol wt 50,000 (12)). Only one of these is titrable with either 2,2’-dipyridyldisulfide or with Ellman’s reagent (la), and the former titrant does not alter the pH dependence of the enzyme spectrum; thus, the functional sulfhydryl group must be shielded from the solvent.

Our previous suggestion (6) that the aldamine was formed by addition of a second amino group to the Schiff base was based primarily on the very small optical activity of the 340 nm ab- sorption band of glutamate decarboxylase. The optical activity of the bound coenzyme probably arises from two sources: The first is the inherent asymmetry of the environment in which the chromophore finds itself, and the second is the chirality of the chromophore itself. The optical activity of reduced glutamate decarboxylase (71 and of enzyme which has been reacted with hydroxylamine (7) show that the asymmetric environment factor

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is appreciable. However, the optical activity of the Schiff base form of the enzyme (H4E& is considerably larger, probably because the carbon-nitrogen bond is slightly out of the plane of the pyridine ring of the coenzyme. Such a structure anticipates the structure of the intermediate which is formed when the en- zyme reacts with substrate. Twisting the carbon-nitrogen bond out of the plane of the ring would lead to optical activity (26). The very small optical activity of the aldamine might be the result of compensating Cotton effects of opposite sign due to environment and due to chirality.

Mechanism of pH J’ump Down-In spite of the very different appearance of the kinetics in the pH jump down and in the pH jump up, the mechanisms of the two reactions are effectively the same, with different rate-determining steps. The intercon- version of the two equilibrium forms of glutamate decarboxylase requires three steps, formation or decomposition of an aldamine (and the associated loss or gain of a proton), loss or gain of three protons, and conformation change. In the following discussion we assume that the 3-proton change and the conformation change occur in the same step. It is clear from our kinetic results that these two changes occur together-that is, when one occurs, the other occurs-but it is not clear whether the two occur simultaneously, or whether the proton change occurs rapidly and induces the conformation change. Since we have no information bearing on this point, we have chosen to assume that the two changes occur in the same step.

Scheme 2 shows our conception of the steps in the pH jump reactions. The equilibrium forms of the enzyme are EM (at high pH) and I14E42o (at low pH). Only counterclockwise re- actions around Scheme 2 are observed; the pH jump down occurs via HZ&,, and the pH jump up occurs via HEW

The first step in the pH jump down is a rapid, reversible pro-

rE,,o/ ,“; HE420

/3H+ CONFORMATION

CHANGE

fast I slow

,773H’

I I

H3E340 + j H4E420 ] y’ - t~\~ GENERAL

.-z--- AC,,, Cl- !“‘SC~TA~~~~S

\ I I

Cl H3E340 slow > ciH4E420 I I

SCHEME 2. The mechanism of the pH jump transitions of gluta- mate decarboxylase. The equilibrium forms of the enzyme are shown in boxes.

tonation and conformation change of the aldamine (E& which forms the metastable protonated aldamine (H3E340). The latter intermediate has the same ultraviolet spectrum as Ea4,,, but is in the low pH conformation. This step occurs too rapidly to be observable in the pH jump studies of the reduced enzyme. The principal evidence for the occurrence of this step is the pH de- pendence of the rate of the pH jump down.

The second step in the pH jump down is rate determining. In this step the aldamine in the low pH conformation decomposes to give the equilibrium low pH form, the ketoenamine (H4EtZ0). This step is general acid catalyzed. The role of the general acid is probably to donate a proton to the aldamine sulfur atom, so that aldamine decomposition involves loss of -SH from car- bon, rather than loss of-S-.

The effects of anions on the pH jump down can also be under- stood in terms of Scheme 2. The rate of the pH jump down in- creases with increasing chloride concentration (Fig. 4). Binding of chloride to I-I3E340 increases the rate of conversion* to H4EdZ0. Chloride does not seem to bind to EsdO.

The effect of chloride ion on the rate of aldamine decomposi- tion is principally on one term. The catalytic constants for hydronium ion (kn in Equation 4), acetic acid, and formic acid are affected only slightly by t,he presence of chloride. However, in the presence of chloride, a new term, k,,, appears in Equation 4. This rate constant is independent of pH and buffer concentra- tion, and has the same value in both acetate and formate buffers.

Addition of chloride to acetate buffers or formate buffers provides a new pathway for aldamine decomposition which does not occur in the absence of chloride (Scheme 2). This new path- way might represent an uncatalyzed reaction, a reaction cata- lyzed by water, or a reaction catalyzed by some group of the enzyme. The first possibility is unlikely on chemical grounds because of the magnitude of rate constant kO. By use of the Bronsted plot for acid catalysis we can estimate that the pK, of the catalytic group5 which is responsible for ko, the “uncata- lyzed” reaction, is near 7. Thus, the catalyst cannot be water. More likely, a histidine residue might be near the active site, and the exact position of this histidine relative to the aldamine might depend on the presence or absence of chloride ion; in the presence of chloride, it is near enough to the aldamine to par- ticipate in its decomposition, whereas in the absence of chloride it is too far away.

The binding of anions to glutamate decarboxylase must be rapid, because the effect of chloride on the pH jump is the same, whether the anion has been incubated with the enzyme prior to the pH jump up or not. This is consistent with the results of NMR experiments which show that the exchange between bound and unbound chloride or bromide is rapid (9).

Many anions bind to the low pH conformation of glutamate decarboxylase. The strength of binding of halide ions decreases in the order I- > Br- > Cl- > F-. This order explains both the anion enhancement of the pH jump down and the anion in- hibition of the pH jump up. The order parallels the Hofmeister

4 The observed dissociation constant for chloride ion from HsEtao (Scheme 2) predicts that the observed pK for the first step in the pH jump down should increase by about 1.0 in the presence of 0.1 M chloride. The data in Fig. 2 are consistent with such an increase but do not provide unequivocal evidence for it.

6 This estimate is made uncertain by the approximate nature of the Bronsted plot and by the lack of knowledge of a reliable factor for correction for the forced proximity of the catalytic group to the aldamine. The combined effect of these two factors is an uncertainty of about f2 in the pK,.

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series (27) and parallels the inhibition of acetoacetate decar- boxylase by anions (28).

Mechanism of pH Jump Up-The rate of the pH jump up rises sharply as the final pH increases from 5.6 to 7.0, then nearly levels off from pH 7.0 to pH 8.0. The slope of the plot of log k versus pH shows that prior to or during the rate-determining step, 3 protons are lost. The midpoint of this 3-proton transi- tion is at pH 6.4. No buffer catalysis of this transition is ob- served.

The reduced enzyme undergoes the pH jump at nearly the same rate as does the native enzyme. This indicates that the pH jump reactions of the native enzyme and the reduced enzyme have the same rate-determining step. No covalent bond change is possible for the reduced enzyme, so this rate-determining step must be the conformation change.

Thus, the first step in the pH jump up removes 3 protons from the enzyme and transforms the enzyme from the low pH conformation to t,he high pH conformation. The nature of the intermediate HE420, which is formed as a result of this step, is not known. The spectrum of the intermediate could not be obtained because it occurs after t,he rate-determining step.

The second step in the pH jump up is the step in which the aldamine is formed. No information is available about the exact rate of this step, but it is clear that the step must be faster than the rate of the conformation change, even at pH 8; if this were not so, then buffer catalysis would be observed at pH 8.

Chloride ion has a marked effect on the rate of the pH jump up. At constant final pH, the observed rate approaches zero as the chloride ion concentration is increased. The pH rate profile for the pH jump in the presence of 0.1 M chloride ion is shifted to higher pH, but is otherwise not significantly affected. Both of these observations indicate that the low pH conforma- tion of the enzyme can bind chloride, whereas the high pH corl-

formation cannot.

Acknowledgments-We thank Dr. M. T. Record for much useful assistance with the stopped flow apparatus and Dr. J. T Gerig for a preprint of a useful manuscript.

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Marion H. O'Leary and Walter Brummund, Jr.pH-DEPENDENT CONFORMATION CHANGE

pH Jump Studies of Glutamate Decarboxylase: EVIDENCE FOR A

1974, 249:3737-3745.J. Biol. Chem. 

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