The Replication of the DNA MoleculeAuthor(s): Peter FongSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 52, No. 3 (Sep. 15, 1964), pp. 641-647Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/72614 .

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VOL. 52, 1964 MICROBIOLOGY: P. FONG 641

nitrogen do, but this hydrogenase activity is inhibited by N2. Sediment fractions containing cell walls and membranes but essentially cell-free will fix N25; this frac- tion also contains all the hydrogenase activity.

* Supported in part by National Science Foundation grant GB-483 and National Institutes of Health grants AI 01417-09 and AI 00848.

t Predoctoral trainee on National Institutes of Health training grant 2G-686. 1Jensen, V., Arch. Mikrobiol., 29, 348 (1958). 2 Goerz, R. D., and R. M. Pengra, J. Bacteriol., 81, 568 (1961). 3Umbreit, W. W., R. H. Burris, and J. F. Stauffer, Manometric Techniques (Minneapolis:

Burgess Publishing Co., 1964). 4 Burris, R. H., and P. W. Wilson, in Methods in Enzymology, ed. S. P. Colowick and N. O.

Kaplan (New York: Academic Press, 1957), vol. 4, pp. 355-366. 5 Proctor, M. H., and P. W. Wilson, Arch. Mikrobiol., 32, 254 (1958). 6 Mortenson, L. E., in The Bacteria, ed. I. C. Gunsalus and R. Y. Stanier (New York: Academic

Press, 1962), vol. 3, pp. 119-166. 7 Grau, F. H., and P. W. Wilson, J. Bacteriol., 85, 446 (1963).

THE REPLICATION OF THE DNA MOLECULE

BY PETER FONG

PHYSICS DEPARTMENT AND LABORATORY OF NUCLEAR STUDIES, CORNELL UNIVERSITY

Communicated by Robert R. Wilson, June 5, 1964

The replication of the DNA molecule consists of three processes: the un- winding of the parent molecule, the duplication of the strands, and the re- winding of the two daughter molecules. The three processes could be sequential or simultaneous. The former alternative has the difficulty that the parent molecule must be completely unwound first. This problem has been discussed in another paper' in which the earlier works on unwinding and replication are listed. Once completely unwound, the strands form random coils; then there arises the additional difficulty of organizing the newly formed molecules into the helical shape. The rewinding of the daughter molecules not only has the topological difficulty as in the unwinding process, but also has the difficulty of being a process in the direction of decreasing entropy. The simultaneous mechanism has the advantage that the parent DNA strands do not change much from the original helical shape, thus eliminating the difficulty of reorganizing the daughter molecules. This alternative is adopted in the original suggestion of Watson and Crick,2 which is later expounded by Levinthal and Crane.' The parent molecule and the two daughters are thought to form a configuration of "Y." Replication takes place only at one point (the crotch of "Y"), and the three branches are required to spin in the same direction with the same speed. The mechanism of the coordinated spinning was not given. It was thought that to complete the theory, we had to solve the problem of how an enzyme can make two different kinds of phosphate bonds simultaneously in two oppositely directed chains and convert the chemical energy into forces to drive the necessary rotations of the three helices. In this paper we shall consider the kine- matics of replication, and the dynamics of unwinding and rewinding. The former should provide the groundwork to investigate further the mechanism of enzyme

VOL. 52, 1964 MICROBIOLOGY: P. FONG 641

nitrogen do, but this hydrogenase activity is inhibited by N2. Sediment fractions containing cell walls and membranes but essentially cell-free will fix N25; this frac- tion also contains all the hydrogenase activity.

* Supported in part by National Science Foundation grant GB-483 and National Institutes of Health grants AI 01417-09 and AI 00848.

t Predoctoral trainee on National Institutes of Health training grant 2G-686. 1Jensen, V., Arch. Mikrobiol., 29, 348 (1958). 2 Goerz, R. D., and R. M. Pengra, J. Bacteriol., 81, 568 (1961). 3Umbreit, W. W., R. H. Burris, and J. F. Stauffer, Manometric Techniques (Minneapolis:

Burgess Publishing Co., 1964). 4 Burris, R. H., and P. W. Wilson, in Methods in Enzymology, ed. S. P. Colowick and N. O.

Kaplan (New York: Academic Press, 1957), vol. 4, pp. 355-366. 5 Proctor, M. H., and P. W. Wilson, Arch. Mikrobiol., 32, 254 (1958). 6 Mortenson, L. E., in The Bacteria, ed. I. C. Gunsalus and R. Y. Stanier (New York: Academic

Press, 1962), vol. 3, pp. 119-166. 7 Grau, F. H., and P. W. Wilson, J. Bacteriol., 85, 446 (1963).

THE REPLICATION OF THE DNA MOLECULE

BY PETER FONG

PHYSICS DEPARTMENT AND LABORATORY OF NUCLEAR STUDIES, CORNELL UNIVERSITY

Communicated by Robert R. Wilson, June 5, 1964

The replication of the DNA molecule consists of three processes: the un- winding of the parent molecule, the duplication of the strands, and the re- winding of the two daughter molecules. The three processes could be sequential or simultaneous. The former alternative has the difficulty that the parent molecule must be completely unwound first. This problem has been discussed in another paper' in which the earlier works on unwinding and replication are listed. Once completely unwound, the strands form random coils; then there arises the additional difficulty of organizing the newly formed molecules into the helical shape. The rewinding of the daughter molecules not only has the topological difficulty as in the unwinding process, but also has the difficulty of being a process in the direction of decreasing entropy. The simultaneous mechanism has the advantage that the parent DNA strands do not change much from the original helical shape, thus eliminating the difficulty of reorganizing the daughter molecules. This alternative is adopted in the original suggestion of Watson and Crick,2 which is later expounded by Levinthal and Crane.' The parent molecule and the two daughters are thought to form a configuration of "Y." Replication takes place only at one point (the crotch of "Y"), and the three branches are required to spin in the same direction with the same speed. The mechanism of the coordinated spinning was not given. It was thought that to complete the theory, we had to solve the problem of how an enzyme can make two different kinds of phosphate bonds simultaneously in two oppositely directed chains and convert the chemical energy into forces to drive the necessary rotations of the three helices. In this paper we shall consider the kine- matics of replication, and the dynamics of unwinding and rewinding. The former should provide the groundwork to investigate further the mechanism of enzyme

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642 MICROBIOLOGY: P. FONG PROC. N. A. S.

action, which will not be considered here. The latter provides a mechanism to generate the necessary rotations which does not require the energy supplied by the chemical reactions and therefore simplifies the chemical problem involved.

Experimental evidence for this mechanism of replication is provided by Cairns4 who took microautographs showing replicating DNA's forming a fork, and by Bonhoeffer and Gierer5 who showed that the chromosome grows at only one point.

Kinematics of Replication. -In order to take advantage of the helical shape of the parent DNA strands, the part of the DNA undergoing duplication should have a form not much changed from the original helix, and the free nucleotides to be assembled should come to a position as close as possible to that they would occupy in the newly formed double helices. Thus, we propose the following mechanism.

Consider the molecular configuration at the crotch of the replicating DNA in a plane perpendicular to the helical axis as represented in Figure 1. The helical axis

. 1.Mechanism of replication of DNA.

9.05A

f the parent DNA passes through the point 0. A base pair, say, T-A, is shown FIG. l.-Mechanism of replication of DNA.

of the parent DNA passes through the point 0. A base pair, say, T-A, is showwn schematically with the proper dimensions. The pair anchors on the backbone chains at points P and Q where the C1 atoms of the sugar rings are situated. Ac- cording to the latest DNA model-Model 3 of Langridge et al.6-the radius of the C1 atoms, OP or OQ, is 5.72 A, and the distance between the two C1 atoms of the pair, PQ, is 10.78 A. The circle passing through P and Q represents that of the C1 atoms. The circle representing the cylinder of the DNA is that of the phos- phorus atoms which has a radius of 9.05 A. The parent DNA is below the plane of the paper, the base pair shown being the last unbroken pair of the parent. The two chains of the parent passing through P and Q are defined as the left and the right chains, respectively. The two daughters are above the plane of the paper represented by two tangent circles of radius 9.05 A centered at B1 and B2. The position of B1 and B2 will now be investigated.

Since the C1 atoms at P and Q of the parent are to become the C1 atoms of the daughter helices, the centers B1 and B2 should be at a distance of 5.72 A from Q and P, respectively. This condition, together with the condition that the daughter cylinders be tangent with each other, does not determine the position of B1 and B2 completely. The position shown in the figure is one which shows left-right sym- metry; this configuration is defined as the S configuration. It is possible to satisfy

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VOL. 52, 1964 MICROBIOLOGY: P. FONG 643

the conditions on B1 and B2, even after S suffers an infinitesimal transformation leading to a configuration defined as S + 3S. Besides, there is another configura- tion obtained from S by inversion of the left circle with respect to the right, the center being at B2'. This configuration is not related to S by infinitesimal trans- formation and is denoted by I(S). Furthermore, I(S) may generate other con- figurations by infinitesimal transformation. That is all. We shall consider replication by the S configuration as shown in the figure. The other configurations will be shown to be less likely.

The left chain of the parent connects with the left chain of the left daughter at point P. So is the right at Q. The left chain of the parent cannot connect the right chain of the daughter because the DNA chain is directional. This establishes the sense of turning of the helices, indicated by the direction of the arrows.

The free nucleotides in the solution may form pairs by hydrogen bonding. Thus, for example, a pair A'-T' may be formed in the solution, then drift into the space of the "big groove" of the parent DNA, and come to a position as shown in the figure. This is the position where the four points P, Q, P', Q' form the four cor- ners of a square. In this square configuration, the two pairs are separated by a distance of at least 2.5 A; thus the van der Waal distances will not be violated. Furthermore, because of the complementary nature of the contour lines of T-A and A'-T', the line P'Q' may be made parallel to PQ. For the same reason, only A'-T', not T'-A', C'-G', or G'-C', may pair with T-A. It is possible that molecular configuration and perhaps secondary bonding may make the square configuration favorable, and the big groove may be filled up with the right pairs before replica- tion. Once the square configuration is arrived at, by some mechanism, presumably an enzyme action, the hydrogen bonds between T-A pair and A'-T' pair are trans- ferred to between T-A' pair and A-T' pair by rotating the four nucleotides (treated as rigid bodies) 900 in the plane about their C1 atoms, the new positions of the bases being shown schematically by dotted lines in the figure. Because of the square configuration, the bases A and T' will be in the right bonding distance after rota- tion; so will A' and T. Since only A'-T' may pair with T-A, the new pairs are always in the correct complementary relation. Thus, the original pair has du-

plicated itself into two identical pairs. This should not be difficult to do geometri- cally, also energetically, because the number of hydrogen bonds is conserved. Fur-

thermore, there is enough room for them to rotate without obstructing each other. After rotation, A-T' pair is to become the last bonded pair of the right daughter, and T' is to be connected to the left chain of it; so will T-A' to the left daughter, and A' will be connected to the right chain of it. The connection of T' to the left chain of the right daughter is not difficult because the desired position of the

pair in the daughter helix is that between Q and P" which is not far from that between Q and P' where the new pair A-T' is formed. The distance P'P" is about 4 A and it only takes a rotation of 22? about Q to move the newly formed pair to its desired position in the daughter helix. The orientation of the base and the nucleotide is just right. The same is true for the T-A' pair in the left daughter. Incidentally, the configuration shows that the two daughters are related in such a way that in the plane of the paper their "small grooves" are almost facing each

other, and therefore the two double helices do not fit into each other's grooves and

they contact by their outer radius as we have assumed. (The small deviation

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644 MICROBIOLOGY: P. FONG PROC. N. A. S.

from exact facing, about 440 out of 360?, corresponds to the turn of about one nucleotide (36?). Considering the van der Waal distances between the contacting phosphate groups of the two chains and the slight amount of fitting into each other's grooves due to the turn of one nucleotide, the use of 9.05 A as the contacting radius seems reasonable.)

Once the two newly formed base pairs are attached to the two daughters, the two daughter helices become completely rigid all the way down to the points Q, P" and P, Q", and therefore they cannot rotate about their axes. (We do not consider the translational motion of the three helices in a direction perpendicular to the helical axis because such motion involves large viscous drag.) On the other hand, the bond between P and Q is now broken, and the parent helix is no longer rigid at the points P and Q; it is rigid all the way up to the points U and V where the next base pair (unbroken) is located, being on a plane 3.4 A below the plane of the paper. Therefore, the parent is free to rotate and will rotate by thermal agitation; but it still cannot rotate in the clockwise direction because the chains are stretched tight at P and Q. It can only rotate in the counterclockwise direction, feeding the chains into the daughter helices through the points P and Q. Once a section of the flexible chain is fed to the daughter, the latter becomes free to rotate until the flexible section of the chain is stretched tight. The daughters may rotate in both directions, but even by the random process of thermal agitation alone, a final state may eventually be reached at which all three helices have rotated through 36? in the counterclockwise direction as the arrows show and the points U and V have moved up to P and Q.7 This is the initial state we begin. At this point the helices cannot rotate in the direction of the arrows further but they muay rotate backward. On the other hand, once returned to the initial state, the du- plication process may now be repeated; after the newly formed pairs have attached to the daughters, the latter become rigid and can no longer rotate backward. Thus, forward rotation of 36? of all three helices alternates the duplication of a pair. The macroscopic result is that all three helices rotate in the forward direction of the arrows with the same speed, which is what we set out to explain. As long as the formation of the bonds is irreversible, the replication can proceed only in one direction until the parent is used up, resulting in two separate replicas; it can- not zip backward.

Before we discuss the mlechanisml of ulnwinding and rewindinig by the random process of thermal agitation, a few renmarks will be mlade concerning the duplica- tion miechanismn.

Our discussion is based on the concept that thte free nucleotides have already formed a pair before they enter the duplication process. An obvious alternative is that each nucleotide acts independently. The pair mnechanism has the ad- vantage that the numlber of hydrogen bonds is conserved and also the advantage that the newly formed pairs are almost in the desired position in the daughter helix and can slip into it without difficulty. If each free nucleotide acts independently, the bond of the parent pair has to be broken first, which is energetically unfavorable. After the bond is broken, the two parent nucleotides may turn around. By the time the new pairs are formied, they are oriented at random with respect to their desired position in the daughter helices and we have the additional difficulty of getting them to their right places.

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VOL. 52, 1964 MICROBIOLOGY: P. FONG 645

The pair mechanismn in the square configuration works only in the S configuration, not in its inversion, I(S). In I(S), the free nucleotides must act independently, and therefore the duplication mechanism is less efficient, if not impossible.

In the configurations S + 6S, one of the newly formed pairs will be closer to its desired position but the other will be farther. The latter change is undesirable. Thus, we conclude that the S configuration is the most efficient.

We mentioned in the beginning that in order to organize the newly formed molecule into the helical shape, the part of the old DNA chain undergoing duplica- tion should not be deformed to a great extent from the original helical shape. The deformation in the present mechanism is simply a bend of the single helix about P (or Q) by 112?. Such a bend is not a drastic deformation from the information point of view because it involves only one bit of information. The newly formed pairs in one daughter are related among themselves according to the relation of the nucleotides in the parent strand which is in the helical configuration. When every pair is bent through the same angle, 112?, they still relate among themselves in the helical configuration, and this answers the topological question of why the newly formed DNA in this mechanism is organized into the same kind of double helix as the parent.

The parent chains passing through P and Q suffer a bend of 112?, and one may wonder if the bonding angles in the chain can sustain such a big bend. Actually, the parent chain comes up from below the paper and then comes out of the paper; the bend in space is less drastic than that seen in the projection plane.

The above mechanism is one in which the parent DNA chains are almost in their desired helical shape and the free nucleotides to be assembled are close to their desired positions and orientations in the daughter helices. Furthermore, the bonding distances, the up-and-down and the left-and-right relationships, of the bases in the daughter helices among themselves and between the base and the backbone chains are exactly right. It is difficult to find other mechanisms that meet all these requirements.

The present mechanism, making use of the paired free nucleotides, has the advantage that the relative positions of the sites of bond formation, such as P" and Q", are fixed once and for all. Therefore, one enzyme molecule with a number of fixed active centers fitted to the sites of bond formation may fulfill all the chemical requirements of the synthesis process, even though the two chains synthesized have opposite polarity.

The Dynamics of Unwinding and Rewinding.-We now consider the detailed mechanism of the unwinding of the parent I)NA and the rewinding of the two daugh- ters sketched above qualitatively. Each of the three helices, if not connected, may rotate about its own axis with an average energy of (l/2)kT, where k is the Boltz- mann constant and T is the absolute temperature of the solution in which the DNA lies. This rotational motion has been investigated in the previous paper.l The fluctuation of the angular velocity of rotation may be considered as the result of a random walk process in the momentum space. This in turn gives rise to a random walk process in the configuration space in which the coordinate is the winding angle. In the previous paper, it is shown that the basic step in the random walk in the configuration space is a rotation of 2.88 X 10-2 turns or 10?, and the time required for the basic step is 6.02 X 10-1? sec. The time required to unwind 36? for the

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646 MICROBIOLOGY: P. FONG PROC. N. A. S.

duplication of one base pair by the random walk process is thus

Tu = (36/10)2 X 6.02 X 10-1 = 7.2 X 10-9 sec. (1)

The lower limit of the time to unwind 36? is

Ti = (36/10) X 6.02 X 10-1 = 2.1 X 10-9 sec. (2)

For the present problem, the three helices are not independent of one another; they are connected by a section of flexible chain. Therefore, the random walks

of the three helices are biased, somewhat similar to that discussed in the previous

paper in that the bias is set up in the configuration space, while the physical random

walk takes place in the momentum space. The present problem is, in principle, more complicated than that of the previous paper because the three random walks are connected to one another. In practice, the present problem is much simpler because the required amount of turning 36? is only 3.6 times the basic step 10? and

the effect of statistics is rather small. Furthermore, in the present mechanism the

bias speeds up the unwinding-as the parent DNA unwinds, the loosened part is

picked up partly by the two daughters, thus reducing the chance of the parent DNA to rewind. Thus, the time Tu given by equation (1) is the upper limit of the unwind-

ing time for 36?, or for a base pair. Since the upper limit and the lower limit are

not far from each other, we may take their average 4.6 X 10-9 sec as the unwinding time for a base pair. The rewinding of the daughters through 36? would take a

time of the order of 10-8 sec, even by random walk alone. Very likely the strain of

the bonding angles at P and Q helps to rewind, and the time required for rewind

would be smaller than 10-s sec. If the strain has a large effect, then both unwind-

ing and rewinding would proceed by a time given by equation (2). Taking the un-

winding time for one base pair to be 4.6 X 10-9 sec, the total time required to un-

wind 2 X 105 base pairs in a DNA nolecule is 9.2 X 10-4 sec. The total time re-

quired to rewind the daughter molecules is of the same order of magnitude. Com-

pared with the replication time of 2-3 min, the time required for unwinding and re-

winding is negligibly small and therefore, unwinding and rewinding will not cause

difficulty in the present mechanism of replication. This discussion also shows that

the replication time is almost exclusively spent for the duplication process. The

time is about 10-3 sec per base pair; the DNA has this much time to find the free

nucleotides in the proper position and to form the bonds.

The total time for unwinding in this mechanism is negligibly small compared with

that in complete unwinding.l Consider the DNA of E. coli with 3 X 106 base

pairs.4 The unwinding time in this mechanism would still be negligibly small, while in complete unwinding it would be of the order of 2 days,l nmuch longer than

the generation time.4 Thus, it is unlikely that replication is preceded by complete

unwinding. According to the present mechanism, the replication time should be

directly proportional to the molecular weight, which seems to be the case when we

compare coli with phage.

Single-stranded DNA from sX174 replicates inside bacteria where it exists in

double-stranded form. The mechanism here discussed may still apply. This also

suggests that the present mechanism is a more efficient one than any based on a

single strand.

It may be stated that in order to take advantage of the mechanism of complemen-

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VOL. 52, 1964 BIOCHEMISTRY: HELMREICH AND CORI 647

tary replication, we have to confiront the double difficulty of unwinding and rewind-

ing. The present mechanism is no exception; the parent DNA unwinds exactly No turns, and the daughters rewind exactly No turns, No being the winding number of the DNA molecule. However, the difficulty is reduced to a minimum by rewind-

ing immediately after unwinding so that there is no large number of unwound turns to cause difficulty both in unwinding and rewinding. In the previous paper, it is stated that the unwinding angular velocity w(t) is a monotonically decreasing func- tion of time or of the number of turns unwound. In the present mechanism, the un- wound turns are removed by the rewinding of the daughters so that the unwinding is

operated at all times near the starting point of the @(t) curve, and the average value of @(t) is very close to its maximum value 5(0). Thus, the unwinding time in the

present mechanism is very close to the lower limit T, in the previous paper. Un-

winding in vivo is therefore nmuch faster than in vitro.

The author wishes to thank Professor Philip Morrison for many discussions and criticisms on the manuscript.

1 Fong, P., "The unwinding of the DNA molecule," these PROCEEDINGS, 52, 239 (1964). 2 Watson, J. D., and F. H. C. Crick, Nature, 171, 737, 964 (1953). 3Levinthal, C., and H. R. Crane, these PROCEEDINGS, 42, 436 (1956). 4 Cairns, J., J. Mol. Biol., 6, 208 (1963). Bonhoeffer, F., and A. Gierer, J. Mol. Biol., 7, 534 (1963).

6 Langridge, R., et al., J. Mol. Biol., 2, 38 (1960). 7 It is likely that the daughters are more disposed to rotate in the direction of the arrows than

backward, since the backward rotation may strain the bonding angles of the chains in the daughter helices in the neighborhood of P and Q. Such a bias would speed up the random process.

THE EFFECTS OF pH AND TEMPERATURE ON THE KINETICS OF THE PHOSPHORYLASE REACTION*

BY ERNST HELMREICH AND CARL F. CORI

DEPARTMENT OF BIOLOGICAL CHEMISTRY,

WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS, MISSOURI

Communicated July 16, 1964

The dependence of muscle phosphorylase on 5'-AIMP for activity, absolute in

the case of phosphorylase b and relative in the case of phosphorylase a, has been

explained by an effect of 5'-AMP on the affinity of the enzyme for its substrates,

inorganic P and glycogen.' Each of these substrates in turn influences the affinity of the enzyme for 5'-AMP. Thus, there is a reciprocal interaction between the

binding sites for substrates and cofactor, suggestive of a conformational alteration of the protein.

Two recent papers on this subject have appeared." 4 Madsen' reported results

similar to those described above for the interaction of glucose-l-P and 5'-AMP with

phosphorylase b. He found that increasing the concentr)ation of glucose-l-P results in an increase in the affinity for 5'-AMP and vice versa. Lowry et al.4 found that 5'-

AMP decreased the dissociation constant of each of the substrates of phosphorylase a. Madsen3 also studied the interaction between ATP, a competitive inhibitor5 of 5'-

VOL. 52, 1964 BIOCHEMISTRY: HELMREICH AND CORI 647

tary replication, we have to confiront the double difficulty of unwinding and rewind-

ing. The present mechanism is no exception; the parent DNA unwinds exactly No turns, and the daughters rewind exactly No turns, No being the winding number of the DNA molecule. However, the difficulty is reduced to a minimum by rewind-

ing immediately after unwinding so that there is no large number of unwound turns to cause difficulty both in unwinding and rewinding. In the previous paper, it is stated that the unwinding angular velocity w(t) is a monotonically decreasing func- tion of time or of the number of turns unwound. In the present mechanism, the un- wound turns are removed by the rewinding of the daughters so that the unwinding is

operated at all times near the starting point of the @(t) curve, and the average value of @(t) is very close to its maximum value 5(0). Thus, the unwinding time in the

present mechanism is very close to the lower limit T, in the previous paper. Un-

winding in vivo is therefore nmuch faster than in vitro.

The author wishes to thank Professor Philip Morrison for many discussions and criticisms on the manuscript.

1 Fong, P., "The unwinding of the DNA molecule," these PROCEEDINGS, 52, 239 (1964). 2 Watson, J. D., and F. H. C. Crick, Nature, 171, 737, 964 (1953). 3Levinthal, C., and H. R. Crane, these PROCEEDINGS, 42, 436 (1956). 4 Cairns, J., J. Mol. Biol., 6, 208 (1963). Bonhoeffer, F., and A. Gierer, J. Mol. Biol., 7, 534 (1963).

6 Langridge, R., et al., J. Mol. Biol., 2, 38 (1960). 7 It is likely that the daughters are more disposed to rotate in the direction of the arrows than

backward, since the backward rotation may strain the bonding angles of the chains in the daughter helices in the neighborhood of P and Q. Such a bias would speed up the random process.

THE EFFECTS OF pH AND TEMPERATURE ON THE KINETICS OF THE PHOSPHORYLASE REACTION*

BY ERNST HELMREICH AND CARL F. CORI

DEPARTMENT OF BIOLOGICAL CHEMISTRY,

WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS, MISSOURI

Communicated July 16, 1964

The dependence of muscle phosphorylase on 5'-AIMP for activity, absolute in

the case of phosphorylase b and relative in the case of phosphorylase a, has been

explained by an effect of 5'-AMP on the affinity of the enzyme for its substrates,

inorganic P and glycogen.' Each of these substrates in turn influences the affinity of the enzyme for 5'-AMP. Thus, there is a reciprocal interaction between the

binding sites for substrates and cofactor, suggestive of a conformational alteration of the protein.

Two recent papers on this subject have appeared." 4 Madsen' reported results

similar to those described above for the interaction of glucose-l-P and 5'-AMP with

phosphorylase b. He found that increasing the concentr)ation of glucose-l-P results in an increase in the affinity for 5'-AMP and vice versa. Lowry et al.4 found that 5'-

AMP decreased the dissociation constant of each of the substrates of phosphorylase a. Madsen3 also studied the interaction between ATP, a competitive inhibitor5 of 5'-

This content downloaded from 194.29.185.102 on Mon, 5 May 2014 23:26:00 PMAll use subject to JSTOR Terms and Conditions