Faraday Discuss., 1992, 93, 85-93

Induced-fit Movements in Adenylate Kinases

Georg E. Schulz Institut f i r Organische Chemie und Biochemie der Universitat, Albertstrasse 21,

7800 Freiburg im Breisgau, Germany

Adenylate kinases have an M , around 23 000 which classifies them among the smallest phosphoryl group transferring enzymes. In order to prevent phosphoryl transfer to water, i. e. hydrolysis, these enzymes undergo induced- fit motions on substrate binding and assembleldisassemble their catalytic centres during each reaction cycle. Details of these processes have been derived from several X-ray structure analyses. The disturbance of these analyses by crystal-packing effects is discussed.

Kinases are enzymes that transfer phosphoryl groups, in most cases to hydroxy groups. Since the hydroxy groups of the surrounding water compete strongly for the phosphoryl groups, a kinase has to shield off its catalytic centre.' Water exclusion could be accomplished by placing the transfer path into the molecular centre, as for example observed in glutathione reductase for an electron transfer: but whereas electrons can penetrate a well packed core, phosphoryl groups cannot. They would need a wide channel, as for instance found with the medium-sized porins, which are located in a memb~ane.~ In an aqueous environment, however, a channel structure would require quite a mass of polypeptide. Actually, kinases choose to exclude water through large polypeptide rearrangements? Most kinases contain more than 350 residues, providing enough shielding material for this purpose. Among this enzyme group the nucleoside monophosphate ( NMP-) kinases are exceptionally small and they catalyse the transfer to an anhydride, which is thermodynamically less favourable than the transfer to a hydroxy group. As a consequence, particularly efficient shielding is required, and large relative mass displacements are expected.

Nucleoside Monophosphate Kinases The nucleoside monophosphate (NMP-) kinases catalyse the reaction

Mg2+ N,TP+ N2MP .C- Mg2'+ NIDP+ N2DP where N1 and N2 represent nucleosides. The enzymes accept normal and deoxynucleo- tides. Their main role is the metabolically efficient recovery of the nucleoside monophos- phates which are produced in protein synthesis, for example. The enzymes are also required in all compartments with a high ATP/ADP or GTP/GDP turnover like mitochondria, chloroplasts and the cytosol of muscle cells, where they maintain the thermodynamic equilibrium between the tri- and di-phosphates. The catalysis requires a divalent cation; Mg2+ works best. This cation binds between the p- and y-phosphoryl groups of NITP, forming only minor contacts to the protein.

The covalent and spatial structures of a number of NMP-kinases are now known; the best analysed is the most abundant group of adenylate (AMP-)kinases within the family.' Appreciable data are available for the guanylate kinase group6-8 and some data are available for the uridylate k ina~es ,~ which also function as cytidylate kinases. In general, N,TP stands for ATP. Only the mitochondria1 matrix adenylate kinase uses

85

Publ

ishe

d on

01

Janu

ary

1992

. Dow

nloa

ded

by U

nive

rsity

of

Pitts

burg

h on

31/

10/2

014

21:4

7:35

. View Article Online / Journal Homepage / Table of Contents for this issue

86 Adenylate Kinases

Fig. 1 Stereoview of the Ca backbone of porcine muscle cytosol adenylate kinase (AKl) without substrates as an example for an NMP-kinase. Several residue positions are given. Reproduced

with permission from D. Dreusicke and G. E. Schulz, J. MoZ. Bid., 1988, 199, 361

GTP more efficiently than ATP, the ratio being ca. 10: 1." The adenylate and guanylate kinases are very specific for (d)AMP and (d)GMP, respectively, and less specific with respect to the base of the triphosphates. In uridylate kinase the monophosphate specificity is less pronounced as also (d)CMP is accepted. The turnover numbers of these enzymes are all in the range of 500s-*, Le. they are not slow.

The M, values of the NMP-kinases range from 20 500 (guanylate kinase from yeast) to 26 100 (bovine mitochondria1 adenylate kinase, A m ) . The family can be subdivided into small and large variants. The chainfold of a small variant is given in Fig. 1. The large variants have an additional domain (INSERT) of 38 residues inserted in the middle of the chain, which is involved in the induced-fit movements. The small variants have a chain segment of 11 residues instead. Moreover, the large variants have disordered chain segments at their N- and C-terminal ends," which are most likely not involved in catalysis but in protein targeting. Neglecting these ends, the small and large variants form M , groups with M , = 21 000 * 1000 and 24 000 * 1000, respectively.

The relationships between all NMP-kinases can be ascertained by amino acid sequence comparisons. For detailed alignments, however, the spatial structures have to be consulted in a number of cases.' The small size of these kinases brings them into an M, range which permits complete structure analyses by NMR in solution. Accord- ingly, this enzyme family may become a model case for studying complicated actions in catalysis.

Structure Analyses of NMP-Kinases

Porcine muscle cytosolic adenylate kinase (AK1) was the first family member the structure of which had been solved by amino acid sequence analysis and X-ray diffrac- t i~n. '** '~ It crystallized in two interconvertible crystal forms. One crystal form exists at pH 5.5 where the enzyme is inactive; its structure has been solved with very limited accuracy at 3.3 A resol~tion. '~ The structure in the other crystal form exists at pH 7.7, where the enzyme is active, and has been elucidated in great detail at 2.1 A resolution (Fig. l ) . 1 5

Publ

ishe

d on

01

Janu

ary

1992

. Dow

nloa

ded

by U

nive

rsity

of

Pitts

burg

h on

31/

10/2

014

21:4

7:35

. View Article Online

G. E. Schulz 87

Fig. 2 Sketches highlighting the structure classification of AMP-kinases as well as the motions (arrows) on substrate binding. The INSERT (. - .) and AMP-binding (- - -) domains are marked, the Gly-loop is indicated by a curved line. ( a ) Cytosolic enzyme AK1 without substrate but with a sulfate ion bound to the Gly - l~op . '~ ( b ) Mitochondria1 matrix enzyme AK3 co-crystallized with AMP." A sulfate ion is bound to the Gly-loop. The locations of INSERT in the two crystallo- graphically independent molecules differ slightly. (c) Yeast" and E. coli21922 enzymes co-crystal- lized with both substrates, ATP and AMP, mimicked by Ap,A.Mg2' as identified by exchange with Mn2+ The additional fifth phosphate of Ap,A, which is disordered in the E. coli

enzyme,22 is marked black

In order to find the substrate positions, crystal-soaking experiments with substrates, inhibitors and analogues were performed under extreme conditions in both crystal forms: applying high concentrations and rapidly collecting low-resolution data before the crystals deteriorated.16 These experiments yielded the general locations of phosphates and of AMP (at that time thought to be the ATP site), but also suggested a second nucleotide site that later turned out to be spurious. The spurious low-resolution density had been caused by bad phases and a crystal-form transition that went undetected during data c~l lec t ion .~~ Unfortunately, the spurious X-ray site received support from NMR experiments.'*

In retrospect, the interconvertible crystal forms of AK1 already indicated the confor- mational flexibility of the enzyme and, in particular, they showed that crystal packing may exert some influence on the enzyme conformation because the crucial Gly-loop (see below) moved away from its place in the molecule in order to form a crystal ~ontact . '~ It seems therefore most important to consider and report all packing contacts in crystal structure analyses. Moreover, the approach to find substrate positions by soaking more or less failed for these crystals, because the region of the active centre was occluded by neighbouring molecules, and because ligand binding presumably caused conformational changes, leading to crystal breakage.

As a side-result of the analyses, the observed secondary structure of AK1 was checked against secondary structure predictions that are solely based on amino acid sequences. This test case came out very favourable for the prediction meth~ds , '~ and in fact much better than in later predictions for other proteins. I therefore conclude that the secondary structures of the NMP-kinases are locally determined by the amino acid sequence, thus playing into the hands of the predictors. I also suggest that they are locally determined in order to establish local stability, which is the prerequisite for an enzyme that needs to be conformationally flexible for catalysis but which also has to assume defined structures.

Advances in the analyses of structures and substrate-binding sites of the NMP-kinases came with co-crystals containing substrates and substrate analogues. The second known medium-resolution structure was that of yeast adenylate kinase ligated with P',P5- bis( adenosine-5'-)pentaphosphate ( AKyst : AP,A),~* an inhibitor that connects ATP and AMP by a fifth phosphate and thus mimicks both substrates. A very similar structure was later established for the adenylate kinase from E. coli, AKeco.: AP,A.~'*~~

The covalently symmetric Ap,A (see Fig. 5, later) showed the binding sites of the substrates but did not allow distinction between the ATP and AMP sites. This was

Publ

ishe

d on

01

Janu

ary

1992

. Dow

nloa

ded

by U

nive

rsity

of

Pitts

burg

h on

31/

10/2

014

21:4

7:35

. View Article Online

88 Adenylate Kinases

subsequently accomplished with the structure of yeast guanylate kinase ligated with GMP (GKyst : GMP)' and with bovine mitochondria1 matrix adenylate kinase ligated with AMP (AK3:AMP)." The assignment was supported by finding that Mn2+ was bound as expected between the p- and y-phosphates of ATpo and that the additional fifth phosphate of Ap,A in the high-resolution structure of the E. coli enzyme (AKceo : Ap5A) was disordered? i.e. it did not have a defined place. Further support came from a comparison with the structurally related G-proteins (see below) that were co-crystallized with GDP and GTP-analogues, showing the position of N1TP?3-26

Most unexpected were the observed gross conformational differences between these crystal structures, which are sketched in Fig. 2.27 Obviously, the substrate-free enzyme has an open structure, whereas the enzyme is closed, compact and globular when both substrates are bound.

Domain Movements and Crystal Packing

As was experienced with conformational changes observed in the crystal form transition of the porcine cytosolic AMP-kinase (AKl), crystal packing may well influence the polypeptide conformation, in some instances just by selecting from an ensemble of conformers in solution. In any case the observed differences indicate where a molecule permits movements. Therefore, it is always advisable to know more than one crystal structure of an enzyme.

The observed structures can be classified using the three groups of Fig.2. The substrate-free open structure is not only found with porcine cytosolic AMP-kinase (AKl),', but also with the corresponding enzyme from carp at low resolution in a different packing scheme.28 The enzyme conformation with bound AMP is known from AK3 : AMP co-crystals which contain two complexes per crystallographic asymmetric unit, ie. in different packing environments." There is also a general correspondence of the substrate position, in particular the a-phosphate of N2MP, with GKyst : GMP.798 A detailed comparison with GKyst is not possible because the chainfold of its GMP- binding domain (&sheet) differs grossly from the AMP-binding domain (a-helices) of

The compact globular conformation with both substrates bound is known from the crystal structure of AKyst : Ap5A2' and the crystal structures of two AKeco : Ap,A complexes in different packing environments (two complexes per asymmetric unit) .21*22

Several other structure analyses of AMP-kinases are under way. For a detailed analysis of the movements, the C a backbones of AK1, AK3:AMP

and AKeco : Ap5A were superimposed, showing that the main bodies of the enzymes consisting of the central parallel &sheet and surrounding helices (residues 1-37,68- 130, 142-194 of AK1, see Fig. 1) are very similar, whereas the remaining parts are different?7 These form the AMP-binding domain (residues 38-67 of AK1) which moves on AMP- binding and continues moving on binding both substrates, and domain INSERT (residues 122-159 of AKeco, the small equivalent segment of AK1 consists of residues 131-141), which rotates on binding both substrates. The observed motions are illustrated in Fig. 3.

A closer inspection of the motions showed that insert is a very rigid entity which moves up to distances of 32%i (rotation 92") without being deformed.27 It consists of an irregular P-sheet as depicted in Fig. 4. Its amino acid sequence is very well conserved among the large variants of the AMP-kinase group. No such domain has been detected yet with the GMP- and UMP-kinases. With 38 residues it ranges among the smallest known rigid polypeptide domains. The structure comparisons assigned the hinges of the movement to residues 121-122 and 159-160. The conformational changes at the hinges are not just rotations around two single bonds, but a combination of numerous rotations. The observation of differing INSERT positions in AK3 : AMP (relative rotation 9") and in particular differing INSERT positions even in the compact AKeco:Ap,A (relative rotation 4") demonstrates clearly the flexibility at the hinge.27 Note that the different INSERT positions are not different rotational states around one common axis.

Publ

ishe

d on

01

Janu

ary

1992

. Dow

nloa

ded

by U

nive

rsity

of

Pitts

burg

h on

31/

10/2

014

21:4

7:35

. View Article Online

G. E. Schulz 89

AMP

AMP AMP ATP

Fig. 3 Full atom models of AMP-kinases aligned on their main bodies. Top: Comparison between the substrate-free cytosolic enzyme AK1 (left, small variant) and the mitochondria1 matrix enzyme AK3 with bound AMP (right, large variant). The AMP-binding domain moves by up to 8 A starting at the position depicted at the left-hand side. Bottom: Comparison between AK3:AMP (left, rotated by 90" around the vertical axis with respect to the model above) and the E. coli enzyme with both substrates bound (right, ATP and AMP are mimicked by inhibitor Ap,A). The AMP-binding domain moves by up to another 8 A, the INSERT domain moves by up to 32 A. Reproduced with permission from G. E. Schulz, C. W. Miiller and K. Diederichs, J. Mol. BioL,

1990,213, 627

Publ

ishe

d on

01

Janu

ary

1992

. Dow

nloa

ded

by U

nive

rsity

of

Pitts

burg

h on

31/

10/2

014

21:4

7:35

. View Article Online

90 Adenylate Kinases

Fig. 4 The Ca backbone structure of domain INSERT consisting of 38 residues (AK3 numbering). This domain behaves like a rigid body during the large induced-fit movements. Reproduced with

permission from K. Diederichs and G. E. Schulz, J. MoZ. Bid., 1991, 217, 541

In contrast to the INSERT motions, those of the AMP-binding domain are not rigid-body movements, but rotations coupled with shearing. They are also smaller, giving rise to backbone displacements of up to 13 A. No clear hinges can be as~igned.~’

Structural Changes during Catalysis Substrate binding to the polypeptide as derived from AKeco : Ap,A is sketched in Fig. 5. The adenosine of AMP is well encapsulated by the AMP-binding domain, which explains the observed high specificity for N2MP. In contrast, the base of NITP is located at a small depression of the molecular surface, in agreement with the low specificity found for this nucleotide. The phosphates are most tightly bound. They are held by five arginines, one lysine and all peptide nitrogens of residues 18-23 of AKl (see Fig. 1, the corresponding residues of AKeco are 10-15), the Gly-loop forming a giant anion hole.29 There is no tight binding between the substrates and residues of the INSERT domain except for Arg-132 and Arg-138 (AKl numbering throughout) that are close to the hinge of INSERT. These two arginines are fixed by salt bridges to Asp-141 and Asp-140 (also near to the hinge). Among the other three arginines, Arg-97 and Arg-149 belong to the main body of the enzyme and Arg-44 to the AMP-binding domain. The Gly-loop, Lys-21 and all five arginines are conserved throughout the enzyme family.

During catalysis the y-phosphoryl group of NITP is transferred to the a-phosphate of N2MP. The structure of AKeco:Ap,A shows that these two groups are in an orientation and location permitting in-line transfer through a bipyramidal pentavalent phosphate intermediate.22 Presumably, Arg-132, Arg-138 and Arg-149 (AK1 numbering) stabilize this intermediate, Mg2+ polarizes the y-phosphoryl group of NITP, facilitating the nucleophilic attack on the phosphorus, Lys-21 moves together with the transferred phosphoryl group as a closely attached companion, while the Gly-loop stabilizes the developing negative charge on the P-phosphate of N,TP (Fig. 5 ) .

The comparison between open and closed structures (Fig. 3) showed that only in the closed structure are the crucial Arg-132 and Arg-138 fixed by the aspartates. The natural conformational state of Arg-149 has not been clarified because it is obviously disturbed by the additional fifth phosphate of AP,A.*~ This suggests the following scenario for substrate binding: On binding the monophosphate, the N2MP-binding

Publ

ishe

d on

01

Janu

ary

1992

. Dow

nloa

ded

by U

nive

rsity

of

Pitts

burg

h on

31/

10/2

014

21:4

7:35

. View Article Online

G. E. Schulz 91

AMP AIP

Gly-loop

Fig. 5 Sketch of the polar main chain contacts (- - - ) between the five phosphates of Ap,A (circles) and AMP-kinase from E. coli.22 The additional fifth phosphate is cross-hatched, assigning the substrates ATP (left) and AMP (right). The Gly-loop is depicted as a series of squares representing residues 18-23 (AKl numbering, see Fig. 1, residues 10-15 in AKeco) running clockwise. The second and third glycine as well as the lysine of the sequence fingerprint are hatched, the lysine sidechain is shown. Except for the €-amino group all interactions are with the peptide nitrogens of the Gly-loop. Arginines are denoted by rectangles with positive charges. Arg-132, Arg-138, Arg-149, Arg-44 and Arg-97 (numbers 123, 156, 167, 36 and 88 in AKeco) are depicted clockwise. Arg-132 and Arg-138 form salt bridges to Asp-141 (left) and Asp-140 (numbers 159 and 158 in AKeco) given by rectangles with negative charges, respectively. The residues are

conserved in the AMP-kinases

domain moves to some extent, but the a-phosphate of N2MP remains rather mobile. On additional binding of NITP, Arg-132 and Arg-138 (located in the INSERT domain near the hinge) are attracted, causing the closing down of INSERT and thus deformations around Asp-140 and Asp-141 at the hinge that lead to the salt bridges (Fig. 5) that are required for catalysis. Accordingly, the enzyme assembles and disassembles its very catalytic centre, i.e. the arginines stabilizing the transition state, during each catalytic cycle of ca. 2ms duration. Concomitantly, the enzyme runs through large domain motions within the cycle. The extent of the immense displacement of INSERT indicated in Fig. 3 and 4 may be a crystal-packing artefact, however, because only part of this motion is required for permitting substrate binding.

Catalytic Constants and Induced Fit of Adenylate Kinases

A number of AMP-kinase mutants, in particular from the E. coli enzyme, have been produced and kinetically analysed. In general, the results support the established location of the active centre.30 The observed changes in the Michaelis constants K,(ATP), K,(AMP) and the maximum velocity V,,, classify the mutants into three groups: Class-I mutations increase both K, values, class I1 decreases V,,, drastically and class I11 increases &(AMP).** Classes I1 and I11 can be understood, because the mutations are located at the transferred phosphoryl group and at the AMP-binding site, respectively. The class I mutations are more complicated as they are located at the ATP-binding site, but they affect the K , values of both substrates.

Class I mutants can now be explained by assuming an equilibrium between two enzyme conformers, Einact, which does not bind substrates and E,, that does, where the mutations shift the distribution towards Einact . Structurally, Einact and E,,, represent the enzyme before and after the full formation of the active centre, which occurs simultaneously with the induced-fit motions. We therefore conclude that the class I mutants disturb the induced fit much more than they disturb the binding of ATP.

Publ

ishe

d on

01

Janu

ary

1992

. Dow

nloa

ded

by U

nive

rsity

of

Pitts

burg

h on

31/

10/2

014

21:4

7:35

. View Article Online

92 Adenylate Kinases

CIassical NTP-binding Fold and Related Proteins It was recognized early on that the Gly-loop that forms the giant anion hole for accommodating the P- and a-phosphates of NtTP in the adenylate kina~es’~.~’ can also be found in F1-ATPa~e31*32 and myosin.33 The Gly-loop is the main part of the characteris- tic sequence fingerprint Gly-X-X-Gly-X-Gly-Lys, which is now known to occur in a large number of NTP-binding and NTP-processing proteins, indicating that it is an important structural feat~re.3~ In the structurally known cases the Gly-loop is located between the first P-strand and the following a-helix of a chain fold consisting of a total of four P-strands and four surrounding helices. This chain fold is essentially the main body of the NMP-kinases and of the G-proteins H-ras-p21 and elongation factor Tu. It is also observed in the more distantly related proteins flavodoxin, GheY and y,S-resolvase. I call it the classical NTP-binding fold.34

Finding a sequence fingerprint in a characteristic chain fold is usually taken as clear indication for a relationship by divergent evolution. For the NTP-binding proteins, however, the characteristic chain fold is small and therefore not very significant; it constitutes only the central part of otherwise different chain folds. Considering the whole chain folds we find sequence conservation at the Gly-loop over times where the chain folds changed appreciably, which is not at all common. Still, divergent evolution within this protein group was recently confirmed by the structure of a protein kina~e,~’ which showed similar NTP binding in a completely unrelated chain fold. Here, the p- and a -phosphates bind at a glycine-rich loop, connecting neighbouring antiparallel p-strands, while the (strongly conserved) lysine binding to the p- and y-phosphates comes from a position distant along the chain. This seems to be a clear case of convergent evolution that demonstrates how important the giant anion hole and the lysine are for phosphoryl transfer.

A closer inspection of the two known high-resolution structures of Gly-loops in NTP-binding proteins22924 showed that the second and third glycines of the fingerprint assume main-chain dihedral angles that are clearly fobidden for residues with side chains. The corresponding angles of the first glycine, though forbidden, are near to the allowed extended chain region.22 It is therefore peculiar that just this first glycine is particularly well conserved, while for instance the second glycine (strongly forbidden) is an alanine in the G-protein elongation factor Tu. Taken together, this peculiarity, the strong conservation of the combination of giant anion hole and lysine, the large induced-fit movement of the AMP-kinases and the observation that the Gly-loop can indeed move in a crystal-form transition of AK1, indicate that dynamical rather than static properties render this structural feature so important. While one particular static structure, e.g. for NTP binding, can be built up in many ways, it is much more difficult to construct a polypeptide that assumes a series of dynamically related structures, as the Gly-loop obviously does. This rationalizes the extreme conservation of the Gly-loop.

I thank Drs. E. Schiltz and A. G. Tomasselli for discussions, and numerous students for their basic work at our institute as reported in the publications.

References 1 W. P. Jencks, Ada Enzymol., 1975,43, 219. 2 P. A. Karplus and G. E. Schulz, J. Mot. Biol., 1989, 210, 163, 3 M. S. Weiss, U. Abele, J. Weckesser, W. Welte, E. Schiltz and G. E. Schulz, Science, 1991, 254, 1627. 4 R. C. McDonald, T. A. Steitz and D. M. Engelman, Biochemistry, 1979, 18, 338. 5 G. E. Schulz, Cold Spring Harbor Symp. Quant. Biol., 1987, 52, 428. 6 A. Berger, E. Schiltz and G. E. Schulz, Eur. J. Biochem., 1989, 184, 433. 7 T. Stehle and G. E. Schulz, J . Mol. Biol., 1990, 211, 249. 8 T. Stehle and G. E. Schulz, J . Mol. Biol., in the press.

Publ

ishe

d on

01

Janu

ary

1992

. Dow

nloa

ded

by U

nive

rsity

of

Pitts

burg

h on

31/

10/2

014

21:4

7:35

. View Article Online

G. E. Schulz 93

9 P. Liljelund, A. Sanni, J. D. Friesen and F. Lacroute, Biochem. Biophys. Res. Commun., 1989, 165,464. 10 A. G. Tomasselli and L. H. Noda, Eur. J. Biochem., 1979, 93, 263. 11 K. Diederichs and G. E. Schulz, J. Mol. BioL, 1991,217, 541. 12 A. Heil, G. Muller, L. H. Noda, T. Pinder, R. H. Schirmer and 1. von Zabern, Eur. J . Biochem., 1974,

13 G. E. Schulz, M. Elzinga, F. Marx and R. H. Schirmer, Nature (London), 1974, 250, 120. 14 D. Dreusicke and G. E. Schulz, J. Mol. Biol., 1988, 203, 1021. 15 D. Dreusicke, P. A. Karplus and G. E. Schulz, J. Mol. Biol., 1988, 199, 359. 16 E. F. Pai, W. Sachsenheimer, R. H. Schirmer and G. E. Schulz, J. Mol. Biol., 1977, 114, 37. 17 K. Diederichs and G. E. Schulz, Biochemistry, 1990,29, 8138. 18 D. C. Fry, D. M. Byler, H. Susi, E. M. Brown, S. Kuby and A. S . Mildvan, Biochemistry, 1988,27,3588. 19 G. E. Schulz, C. D. Barry, J. Friedman, P. Y. Chou, G. D. Fasman, A. V. Finkelstein, V. I. Lim, 0. B.

Ptitsyn, E. A. Kabat, T. T. Wu, M. Levitt, B. Robson and K. Nagano, Nature (London), 1974,250,140. 20 U. Egner, A. G. Tomasselli and G. E. Schulz, J. Mol. BioL, 1987, 195, 649. 21 C. W. Muller and G. E. Schulz, J. Mol. BioL, 1988,202,909. 22 C. W. Muller and G. E. Schulz, J. Mol. Biol., 1992, 224, 159. 23 A. M. deVos, L. Tong, M. V. Milburn, P. M. Matias, J. Jancarik, S. Noguchi, S. Nishimura, K. Miura,

24 E. F. Pai, U, Krengel, G. A. Petsko, R. S. Goody, W. Kabsch and A. Wittinghofer, EMBO J., 1990,9,

25 F. Jurnak, Science, 1985, 230, 32. 26 B. F. C. Clark, M. Kjeldgaard, T. F. M. LaCour, S. Thirup and J. Nyborg, Biochim. Biophys. Acta,

27 G. E. Schulz, C. W. Muller and K. Diederichs, J . Mol. Biol., 1990, 213, 627. 28 C. Reuner, M. Hable, M. Wilmanns, E. Kiefer, E. Schiltz and G. E. Schulz, Protein Seq. Data Anal.,

29 D. Dreusicke and G. E. Schulz, FEBS Lett., 1986, 208, 301. 30 M-D. Tsai and H. Yan, Biochemistry, 1991,30,6806. 31 J. E. Walker, M. Saraste, M. J. Runswick and N. J. Gay, EMBO J., 1982, 8, 945. 32 P. D. Vogel and R. L. Cross, J. Biol. Chem., 1991,266, 6101. 33 J. Karn, S. Brenner and L. Barnett, Proc. Natl. Acud. Sci. USA, 1983, 80, 4253. 34 G. E. Schulz, Curr. Opinion Struct. Biol., 1991, 2, 61. 35 D. R. Knighton, J. Zheng, L. F. TenEyck, V. A. Ashford, N-H. Xuong, S. S. Taylor and J. M. Sowadski,

43, 131.

E. Ohtsuka and S-H. Kim, Science, 1988, 239, 888.

2351.

1990, 1050,203.

1988, 1, 335.

Science, 1991, 253, 407.

Paper 2/00193D; Received 14th January, 1992

Publ

ishe

d on

01

Janu

ary

1992

. Dow

nloa

ded

by U

nive

rsity

of

Pitts

burg

h on

31/

10/2

014

21:4

7:35

. View Article Online