19o PRELIMINARY NOTES

PN I 0 0 0 ~ Amidase f rom Pseudomonas aeruginosa: a mult i -headed enzyme

I t has been previously reported 1 that Pseudomonas aeruginosa 86o2/A, during the later stages of growth on acetate as sole carbon source, forms large quantities of an enzyme which is specific s for the hydrolysis of several aliphatic amides (Reaction I) :

R 'C O N H ~ + H O H + R ' C O O H + N H a (I)

Sonic extracts of the organism rich in this enzyme have been found to be capable also of effecting the transfer of the acyl moiety of such amides to hydroxylamine (Reaction 2):

R ' C O N H 2 + NH2OH -~ R ' C O N H O H + NH 3 (2)

The rate of Reaction I with propionamide was 2.6 times the rate observed with acetamide as substrate. However, the relative rates of Reactions i : 2 with propiona- mide were in the proportion 4.3 : I whereas, with acetamide, they were in the proportion I : 4-3.

These relative proportions of enzymic activities were maintained constant throughout 35-fold purification of the extract (Table I), which suggests that both enzymic reactions are catalysed either by one enzyme, or by two enzymes so tightly associated as to remain together in constant proportion throughout the purification procedure. Since the addition of cyanoacetamide to the growth medium, which has been shown to abolish or arrest the formation of the amidase 1, also abolishes or arrests the formation of the enzyme catalysing Reaction 2, it is likely that the two enzymic reactions observed are catalysed by one enzyme.

TABLE I

E N Z Y M I C A C T I V I T I E S T H R O U G H O U T P U R I F I C A T I O N

Amidase act ivi ty (Reaction I) was determined as previously described 2. Transferase (Reaction 2) was assayed as the rate of hyd roxama te format ion a t 37 ° f rom propionamide (3oo/zmoles) in the presence of 25o #moles of po tass ium phospha te (pH 7.2), io/~moles of cysteine • HC1, 30o/zmoles of freshly-neutralized hydroxy lamine . HC1, enzyme and water to o. 5 ml. The reaction was s topped af te r 2 - i o rain by addit ion of 1. 5 ml of IO% FeC1 a in 3.3% trichloroacetic acid and o.66 N HC1, protein was removed by centfifugation and the hyd roxama te colour was measured at 54 ° m/z

against a solution containing all the reagents except enzyme.

Step Material Total Specific activity t O[

Amidase ( , ) protein A midase Transferase Ratio : Tranfferase (2)

(rag) (Reaction i ) (Reaction 2)

i Sonic extract 486o 14.7 3.34 4-4 2 Heat - t rea ted ext rac t 1568 43.2 9.9o 4-4 3 Dialysed heat- t reated ext rac t 1525 42.0 9.78 4.3 4 Combined fractions af te r

DEAE-cellulose chromatog- r aphy 14o.5 154 34 .1 4.5

5 After fract ionat ion wi th am- mon ium sulphate 4o.2 31o 61.3 5.0

6 Eluate from alumina Cy gel lo.8 515 12o 4.3

* Specific activities are expressed as / ,moles of N H 3 liberated (amidase) or hyd roxama te formed (transferase) f rom propionamide per mg of soluble protein per min.

Biochim. Biophys. Acta, 64 (1962) t9o- I91

PRELIMINARY NOTES I 9 I

Although the enzyme was not resolw~d with respect to possible individual pro- teins, the use of inhibitors enabled it to be resolved with respect to the individual enzymic activities (Fig. I). Thus, urea at low concentrations was a powerful inhibitor of amidase activity (Reaction i) but exerted a negligible effect on transferase activity (Reaction 2) : at a concentration of 2- IO -3 M urea and using propionamide as sub- strate, the transferase activity of the purified enzyme was identical with that ob- served in the absence of urea, though no amidase activity was detected (Fig. IA). Conversely, the addition of KF affected transferase activity more powerfully than amidase activity: in the presence of 0.i M KF, the amidase activity was 96% of that in the absence of fluoride whereas the transferase activity had been reduced to

100,

2C

i i

10-5 10-'4 10-3 10 -2 10 -1

60

35

"~ ac

:i

I8°/o (Fig. IB).

1oo[

6O

40

2O

. j i

1 10 -a 10 -2 10 - I

Concent ra t ion (M) of inh ib i to r

Fig. I. Effect of urea (A) and K F (B) on the amidase (©) and transferase (2x) activity of the purified enzyme. Propionamide was used as subs t ra te th roughout . Details of the assays used are

given in Table I.

These results indicate that the two enzymic reactions observed are catalysed at different sites. If, as seems likely, they are indeed brought about by one enzyme, this enzyme must be regarded as a 'multi-headed' enzyme in the sense of RACKER AND KRIMSKY'S definition 3 of "a protein which contains more than one kind of catalytic site on the same molecule". They further indicate that the inducible amidase of Ps. aeruginosa differs from the similar, though less active, enzyme re- ported to be present in myeobacteria 4, in which amidase and transferase activities appear to be. readily resolvable.

We thank Miss E. SEAMAN for technical assistance, and gratefully acknowledge support from the Medical Research Council, under a Grant for Scientific Assistance, and from the Air Force Office of Scientific Research, OAR, through the European Office, Aerospace Research, U.S. Air Force, under grant AF EOAR 61-12.

Department of Biochemistry, University of Leicester,

Leicester (Great Britain)

M. KELLY H. L. KORNBERG

1 M. KELLY AND H. L. KORNBERG, Biochim. Biophys. Acta, 59 (1962) 517. 2 M. KELLY AND P. H. CLARKE, J. Gen. Microbiol., 27 (I962) 305. 3 E. RACKER AND I. KRIMSKY, Federation Proc., 17 (1958) 1135. 4 T. KIMURA, J. Biochem. (Tokyo), 46 (1959) I27I.

Received July 23rd, 1962

Biochim. Biophys. Acta, 64 (1962) 19o-191