Biomedical subjects

R B Honzatko

Publications and source records attributed to R B Honzatko.

At least 37 records · Page 2Linked to original sources

PubMed1998

Identification of a phosphate regulatory site and a low affinity binding site for glucose 6-phosphate in the N-terminal half of human brain hexokinase.

Crystal structures of human hexokinase I reveal identical binding sites for phosphate and the 6-phosphoryl group of glucose 6-phosphate in proximity to Gly87, Ser88, Thr232, and Ser415, a binding site for the pyranose moiety of glucose 6-phosphate in proximity to Asp84, Asp413, and Ser449, and a single salt link involving Arg801 between the N- and C-terminal halves. Purified wild-type and mutant enzymes (Asp84 --> Ala, Gly87 --> Tyr, Ser88 --> Ala, Thr232 --> Ala, Asp413 --> Ala, Ser415 --> Ala, Ser449 --> Ala, and Arg801 --> Ala) were studied by kinetics and circular dichroism spectroscopy. All eight mutant hexokinases have kcat and Km values for substrates similar to those of wild-type hexokinase I. Inhibition of wild-type enzyme by 1,5-anhydroglucitol 6-phosphate is consistent with a high affinity binding site (Ki = 50 microM) and a second, low affinity binding site (Kii = 0.7 mM). The mutations of Asp84, Gly87, and Thr232 listed above eliminate inhibition because of the low affinity site, but none of the eight mutations influence Ki of the high affinity site. Relief of 1,5-anhydroglucitol 6-phosphate inhibition by phosphate for Asp84 --> Ala, Ser88 --> Ala, Ser415 --> Ala, Ser449 --> Ala and Arg801 --> Ala mutant enzymes is substantially less than that of wild-type hexokinase and completely absent in the Gly87 --> Tyr and Thr232 --> Ala mutants. The results support several conclusions. (i) The phosphate regulatory site is at the N-terminal domain as identified in crystal structures. (ii) The glucose 6-phosphate binding site at the N-terminal domain is a low affinity site and not the high affinity site associated with potent product inhibition. (iii) Arg801 participates in the regulatory mechanism of hexokinase I.

Binding Sites↗

PubMed1998

Directed mutations in the poorly defined region of porcine liver fructose-1,6-bisphosphatase significantly affect catalysis and the mechanism of AMP inhibition.

Asn64, Asp68, Lys71, Lys72, and Asp74 of porcine liver fructose-1, 6-bisphosphatase (FBPase) are conserved residues and part of a loop for which no electron density has been observed in crystal structures. Yet mutations of the above dramatically affect catalytic rates and/or AMP inhibition. The Asp74 --> Ala and Asp74 --> Asn mutant enzymes exhibited 50,000- and 2,000-fold reductions, respectively, in kcat relative to wild-type FBPase. The pH optimum for the catalytic activity of the Asp74 --> Glu, Asp68 --> Glu, Asn64 --> Gln, and Asn64 --> Ala mutant enzymes shifted from pH 7.0 (wild-type enzyme) to pH 8.5, whereas the Lys71 --> Ala mutant and Lys71,72 --> Met double mutant had optimum activity at pH 7.5. Mg2+ cooperativity, Km for fructose 1,6-bisphosphate, and Ki for fructose 2,6-bisphosphate were comparable for the mutant and wild-type enzymes. Nevertheless, for the Asp74 --> Glu, Asp68 --> Glu, Asn64 --> Gln, and Asn64 --> Ala mutants, the binding affinity for Mg2+ decreased by 40-125-fold relative to the wild-type enzyme. In addition, the Asp74 --> Glu and Asn64 --> Ala mutants exhibited no AMP cooperativity, and the kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive to noncompetitive. The double mutation Lys71,72 --> Met increased Ki for AMP by 175-fold and increased Mg2+ affinity by 2-fold relative to wild-type FBPase. The results reported here strongly suggest that loop 51-72 is important for catalytic activity and the mechanism of allosteric inhibition of FBPase by AMP.

Adenosine Monophosphate↗

PubMed1998

Ambiguities in mapping the active site of a conformationally dynamic enzyme by directed mutation. Role of dynamics in structure-function correlations in Escherichia coli adenylosuccinate synthetase.

On the basis of ligated crystal structures, Asn21, Asn38, Thr42, and Arg419 are not involved in the chemical mechanism of adenylosuccinate synthetase from Escherichia coli, yet these residues are well conserved across species. Purified mutants (Asp21 --> Ala, Asn38 --> Ala, Asn38 --> Asp, Asn38 --> Glu, Thr42 --> Ala, and Arg419 --> Leu) were studied by kinetics, circular dichroism spectroscopy, and equilibrium ultracentrifugation. Asp21 and Arg419 are not part of the active site, yet mutations at positions 21 and 419 lower kcat 20- and 10-fold, respectively. Thr42 interacts only through its backbone amide with the guanine nucleotide, yet its mutation to alanine significantly increases Km for all substrates. Asn38 hydrogen-bonds directly to the 5'-phosphoryl group of IMP, yet its mutation to alanine and glutamate has no effect on Km values, but reduces kcat by 100-fold. The mutation Asn38 --> Asp causes 10-57-fold increases in Km for all substrates along with a 30-fold decrease in kcat. At pH 5.6, however, the Asn38 --> Asp mutant is more active, yet binds IMP 100-fold more weakly, than the wild-type enzyme. Proposed mechanisms of ligand-induced conformational change and subunit aggregation can account for the properties of mutant enzymes reported here. The results underscore the difficulty of using directed mutations alone as a means of mapping the active site of an enzyme.

Adenylosuccinate Synthase↗

PubMed1998

The mechanism of regulation of hexokinase: new insights from the crystal structure of recombinant human brain hexokinase complexed with glucose and glucose-6-phosphate.

BACKGROUND: Hexokinase I is the pacemaker of glycolysis in brain tissue. The type I isozyme exhibits unique regulatory properties in that physiological levels of phosphate relieve potent inhibition by the product, glucose-6-phosphate (Gluc-6-P). The 100 kDa polypeptide chain of hexokinase I consists of a C-terminal (catalytic) domain and an N-terminal (regulatory) domain. Structures of ligated hexokinase I should provide a basis for understanding mechanisms of catalysis and regulation at an atomic level. RESULTS: The complex of human hexokinase I with glucose and Gluc-6-P (determined to 2.8 A resolution) is a dimer with twofold molecular symmetry. The N- and C-terminal domains of one monomer interact with the C- and N-terminal domains, respectively, of the symmetry-related monomer. The two domains of a monomer are connected by a single alpha helix and each have the fold of yeast hexokinase. Salt links between a possible cation-binding loop of the N-terminal domain and a loop of the C-terminal domain may be important to regulation. Each domain binds single glucose and Gluc-6-P molecules in proximity to each other. The 6-phosphoryl group of bound Gluc-6-P at the C-terminal domain occupies the putative binding site for ATP, whereas the 6-phosphoryl group at the N-terminal domain may overlap the binding site for phosphate. CONCLUSIONS: The binding synergism of glucose and Gluc-6-P probably arises out of the mutual stabilization of a common (glucose-bound) conformation of hexokinase I. Conformational changes in the N-terminal domain in response to glucose, phosphate, and/or Gluc-6-P may influence the binding of ATP to the C-terminal domain.

Adenosine Triphosphate↗

PubMed1998

The roles of glycine residues in the ATP binding site of human brain hexokinase.

Mutants of hexokinase I (Arg539 --> Lys, Thr661 --> Ala, Thr661 --> Val, Gly534 --> Ala, Gly679 --> Ala, and Gly862 --> Ala), located putatively in the vicinity of the ATP binding pocket, were constructed, purified to homogeneity, and studied by circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and initial velocity kinetics. The wild-type and mutant enzymes have similar secondary structures on the basis of CD spectroscopy. The mutation Gly679 --> Ala had little effect on the kinetic properties of the enzyme. Compared with the wild-type enzyme, however, the Gly534 --> Ala mutant exhibited a 4000-fold decrease in kcat and the Gly862 --> Ala mutant showed an 11-fold increase in Km for ATP. Glucose 6-phosphate inhibition of the three glycine mutants is comparable to that of the wild-type enzyme. Inorganic phosphate is, however, less effective in relieving glucose 6-phosphate inhibition of the Gly862 --> Ala mutant, relative to the wild-type enzyme and entirely ineffective in relieving inhibition of the Gly534 --> Ala mutant. Although the fluorescence emission spectra showed some difference for the Gly862 --> Ala mutant relative to that of the wild-type enzyme, indicating an environmental alteration around tryptophan residues, no change was observed for the Gly534 --> Ala and Gly679 --> Ala mutants. Gly862 --> Ala and Gly534 --> Ala are the first instances of single residue mutations in hexokinase I that affect the binding affinity of ATP and abolish phosphate-induced relief of glucose 6-phosphate inhibition, respectively.

Adenosine Triphosphate↗

PubMed1998

Mutations to alter Aspergillus awamori glucoamylase selectivity. II. Mutation of residues 119 and 121.

Mutations Ser119-->Glu, Ser119-->Gly, Ser119-->Trp, Gly121-->Ala and Gly121-->Ala/Ser411-->Gly were constructed in glucoamylase to change substrate specificity. Mutation Ser411-->Gly was already known to decrease glucoamylase selectivity toward isomaltose formation and to increase peak glucose yield. All mutated glucoamylases had slightly lower specific activities on maltose than on wild-type glucoamylase. Ser119-->Glu, Ser119-->Gly and Ser119-->Trp glucoamylases were about as active on isomaltose and DP 4-7 maltooligosaccharides as wild-type glucoamylase. Gly121-->Ala and Gly121-->Ala/Ser411-->Gly glucoamylases were less active. At 55 degrees C Ser119-->Glu, wild-type, Ser119-->Trp, Ser119-->Gly, Gly121-->Ala and Gly121-->Ala/Ser411-->Gly glucoamylases had progressively higher peak glucose yields, generally in the opposite order to their activities. There was also an inverse correlation between peak glucose yield and ratio of initial rate of isomaltose production from glucose condensation to that of glucose production from maltodextrin hydrolysis. The effect of mutations Gly121-->Ala and Ser411-->Gly was not additive in predicting the effect of the double mutation on the ratio or on peak glucose yield.

Aspergillus↗

PubMed1997

Major changes in the kinetic mechanism of AMP inhibition and AMP cooperativity attend the mutation of Arg49 in fructose-1,6-bisphosphatase.

The significance of subunit interface residues Arg49 and Lys50 in the function of porcine liver fructose-1,6-bisphosphatase was explored by site-directed mutagenesis, initial rate kinetics, and circular dichroism spectroscopy. The Lys50 --> Met mutant had kinetic properties similar to the wild-type enzyme but was more thermostable. Mutants Arg49 --> Leu, Arg49 --> Asp, Arg49 --> Cys were less thermostable than the wild-type enzyme yet exhibited wild-type values for kcat and Km. The Ki for the competitive inhibitor fructose 2,6-bisphosphate increased 3- and 5-fold in Arg49 --> Leu and Arg49 --> Asp, respectively. The Ka for Mg2+ increased 4-8-fold for the Arg49 mutants, with no alteration in the cooperativity of Mg2+ binding. Position 49 mutants had 4-10-fold lower AMP affinity. Most significantly, the mechanism of AMP inhibition with respect to fructose 1,6-bisphosphate changed from noncompetitive (wild-type enzyme) to competitive (Arg49 --> Leu and Arg49 --> Asp mutants) and to uncompetitive (Arg49 --> Cys mutant). In addition, AMP cooperativity was absent in the Arg49 mutants. The R and T-state circular dichroism spectra of the position 49 mutants were identical and superimposable on only the R-state spectrum of the wild-type enzyme. Changes from noncompetitive to competitive inhibition by AMP can be accommodated within the framework of a steady-state Random Bi Bi mechanism. The appearance of uncompetitive inhibition, however, suggests that a more complex mechanism may be necessary to account for the kinetic properties of the enzyme.

Adenosine Monophosphate↗

PubMed1997

Relationship of conserved residues in the IMP binding site to substrate recognition and catalysis in Escherichia coli adenylosuccinate synthetase.

Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme. Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP.

Adenylosuccinate Synthase↗

PubMed1997

Entrapment of 6-thiophosphoryl-IMP in the active site of crystalline adenylosuccinate synthetase from Escherichia coli.

Crystal structures of adenylosuccinate synthetase from Escherichia coli complexed with Mg2+, 6-thiophosphoryl-IMP, GDP, and hadacidin at 298 and 100 K have been refined to R-factors of 0.171 and 0.206 against data to 2.8 and 2.5 A resolution, respectively. Interactions of GDP, Mg2+ and hadacidin are similar to those observed for the same ligands in the complex of IMP, GDP, NO3-, Mg2+ and hadacidin (Poland, B. W., Fromm, H. J. & Honzatko, R. B. (1996). J. Mol. Biol. 264, 1013-1027). Although crystals were grown from solutions containing 6-mercapto-IMP and GTP, the electron density at the active site is consistent with 6-thiophosphoryl-IMP and GDP. Asp-13 and Gln-224 probably work in concert to stabilize the 6-thioanion of 6-mercapto-IMP, which in turn is the nucleophile in the displacement of GDP from the gamma-phosphate of GTP. Once formed, 6-thiophosphoryl-IMP is stable in the active site of the enzyme under the conditions of the structural investigation. The direct observation of 6-thiophosphoryl-IMP in the active site is consistent with the putative generation of 6-phosphoryl-IMP along the reaction pathway of the synthetase.

Adenylosuccinate Synthase↗

PubMed1997

Residues essential for catalysis and stability of the active site of Escherichia coli adenylosuccinate synthetase as revealed by directed mutation and kinetics.

Examined here by directed mutation, circular dichroism spectroscopy, and kinetics are the relationships of five residues, Asp13, Glu14, Lys16, His41, and Arg131, to the catalytic function and structural organization of adenylosuccinate synthetase from Escherichia coli. The D13A mutant has no measurable activity. Mutants E14A and H41N exhibit 1% of the activity of the wild-type enzyme and 2-7-fold increases in the Km of substrates. The mutant K16Q has 34% of the activity of wild-type enzyme and Km values for substrates virtually unchanged from those of the wild-type system. Mutation of Arg131 to leucine caused only a 4-fold increase in the Km for aspartate relative to the wild-type enzyme. The dramatic effects of the D13A, E14A, and H41N mutations on kcat are consistent with the putative roles assigned to Asp13 (catalytic base), His41 (catalytic acid), and Glu14 (structural organization of the active site). The modest effect of the R131L mutation on the binding of aspartate is also in harmony with recent crystallographic investigations, which suggests that Arg131 stabilizes the conformation of the loop that binds the beta-carboxylate of aspartate. The modest effect of the K16Q mutation, however, contrasts with significant changes brought about by the mutation of the corresponding lysines in the P-loop of other GTP- and ATP-binding proteins. Crystallographic structures place Lys16 in a position of direct interaction with the gamma-phosphate of GTP. Furthermore, lysine is present at corresponding positions in all known sequences of adenylosuccinate synthetase. We suggest that along with a modest role in stabilizing the transition state of the phosphotransfer reaction, Lys16 may stabilize the enzyme structurally. In addition, the modest loss of catalytic activity of the K16Q mutant may confer such a selective disadvantage to E. coli that this seemingly innocuous mutation is not tolerated in nature.

Adenylosuccinate Synthase↗

PubMed1997

A study of Escherichia coli adenylosuccinate synthetase association states and the interface residues of the homodimer.

The state of aggregation of adenylosuccinate synthetase from Escherichia coli is a point of controversy, with crystal structures indicating a dimer and some solution studies indicating a monomer. Crystal structures implicate Arg143 and Asp231 in stabilizing the dimer, with Arg143 interacting directly with bound IMP of the 2-fold related subunit. Residue Arg143 was changed to Lys and Leu, and residue Asp231 was changed to Ala. Matrix-assisted laser desorption ionization mass spectroscopy and analytical ultracentrifugation of the wild-type and the mutant enzymes indicate a mixture of monomers and dimers, with a majority of the enzyme in the monomeric state. In the presence of active site ligands, the wild-type enzyme exists almost exclusively as a dimer, whereas the mutant enzymes show only slightly decreased dissociation constants for the dimerization. Initial rate kinetic studies of the wild-type and mutant enzymes show similar kcat and Km values for aspartate. However, increases in the Km values of GTP and IMP are observed for the mutant. Changes in dissociation constants for IMP are comparable with changes in Km values. Our results suggest that IMP binding induces enzyme dimerization and that two residues in the interface region, Arg143 and Asp231, play significant roles in IMP and GTP binding.

Adenylosuccinate Synthase↗

PubMed1996

Biochemical properties of mutant and wild-type fructose-1,6-bisphosphatases are consistent with the coupling of intra- and intersubunit conformational changes in the T- and R-state transition.

The significance of interactions between AMP domains in recombinant porcine fructose-1,6-bisphosphatase (FBPase) is explored by site-directed mutagenesis and kinetic characterization of homogeneous preparations of mutant enzymes. Mutations of Lys42, Ile190, and Gly191 do not perturb the circular dichroism spectra, but have significant effects on ligand binding and mechanisms of cooperativity. The Km for fructose 1,6-bisphosphate and the Ki for the competitive inhibitor, fructose 2,6-bisphosphate, decreased by as much as 4- and 8-fold, respectively, in the Q32L, K42E, K42T, I190T, and G191A mutants relative to the wild-type enzyme. Q32L, unlike the other four mutants, exhibited a 1.7-fold increase in Kcat. Mg2+ binding is sigmoidal for the five mutants as well as for the wild-type enzyme, but the Mg2+ affinities were decreased (3-22-fold) in mutant FBPases. With the exception of Q32L (8-fold increase), the 50% inhibiting concentrations of AMP for K42E, K42T, I190T, and G191A were increased over 2,000-fold (>10 mM) relative to the wild-type enzyme. Most importantly, a loss of AMP cooperativity was found with K42E, K42T, I190T, and G191A. In addition, the mechanism of AMP inhibition with respect to Mg2+ was changed from competitive to noncompetitive for K42T, I190T, and G191A FBPases. Structural modeling and kinetic studies suggest that Lys42, Ile190, and Gly191 are located at the pivot point of intersubunit conformational changes that energetically couple the Mg2+-binding site to the AMP domain of FBPase.

Adenosine Monophosphate↗

PubMed1996

Crystal structures of adenylosuccinate synthetase from Escherichia coli complexed with GDP, IMP hadacidin, NO3-, and Mg2+.

Crystal structures of adenylosuccinate synthetase from Esherichia coli complexed with Mg2+, IMP, GDP, NO3- and hadacidin at 298 and 100 K have been refined to R-factors of 0.188 and 0.206 against data to 2.8 A and 2.5 A resolution, respectively. Conformational changes of up to 9 A relative to the unligated enzyme occur in loops that bind to Mg2+, GDP, IMP and hadacidin. Mg2+ binds directly to GDP, NO3-, hadacidin and the protein, but is only five-coordinated. Asp13, which approaches, but does not occupy the sixth coordination site of Mg2+, hydrogen bonds to N1 of IMP. The nitrogen atom of NO3- is approximately 2.7 A from O6 of IMP, reflecting a strong electrostatic interaction between the electron-deficient nitrogen atom and the electron-rich O6. The spatial relationships between GDP, NO3- and Mg2+ suggest an interaction between the beta,gamma-bridging oxygen atom of GTP and Mg2+ in the enzyme-substrate complex. His41 hydrogen bonds to the beta-phosphate group of GDP and approaches bound NO3-. The aldehyde group of hadacidin coordinates to the Mg2+, while its carboxyl group interacts with backbone amide groups 299 to 303 and the side-chain of Arg303. The 5'-phosphate group of IMP interacts with Asn38, Thr129, Thr239 and Arg143 (from a monomer related by 2-fold symmetry). A mechanism is proposed for the two-step reaction governed by the synthetase, in which His41 and Asp13 are essential catalytic side-chains.

Adenylosuccinate Synthase↗

PubMed1996

Refined crystal structure of adenylosuccinate synthetase from Escherichia coli complexed with hydantocidin 5'-phosphate, GDP, HPO4(2-), Mg2+, and hadacidin.

A crystal structure of adenylosuccinate synthetase from Escherichia coli, complexed with 5'-phosphate, GDP, HPO4(2-), Mg2+, and hadacidin at 100 K, has been refined to an Rfactor of 0.195 against data to 2.6 A resolution. Bond lengths and angles deviate from expected values by 0.012 A and 1.86 degrees, respectively. Lys 16 and backbone amides 15-17 and 42 interact with the phosphates of GDP, while Ser 414, Asp 333, and backbone amides 331 and 416 interact with the base. Mg2+ is octahedrally coordinated. Oxygen atoms from GDP, phosphate, and hadacidin define the equatorial plane of coordination of the Mg2+, while backbone carbonyl 40 and the side chain of Asp 13 are the apical ligands. HPO4(2-) hydrogen bonds with Lys 16, His 41, backbone amides 13, 40, and 224, and the base moiety of the hydantocidin inhibitor. The carboxylate of hadacidin interacts with Arg 303 and Thr 301; its N-formyl group coordinates to Mg2+, and its hydroxyl group hydrogen bonds with Asp 13. The 5'-phosphate of the hydantocidin inhibitor interacts with Asn 38, Thr 129, and Thr 239 but is approximately 3.5 A from Arg 143 (related by molecular 2-fold symmetry). The base moiety of hydantocidin 5'-phosphate hydrogen bonds to Gln 224 and participates in a hydrogen-bonded network that includes the phosphate molecule, several water molecules, and Asp 13. Hydantocidin 5'-phosphate, GDP, HPO4(2-), and Mg2+ may represent a set of synergistic inhibitors even more effective than the combination of IMP, GDP, NO3-, and Mg2+.

Adenylosuccinate Synthase↗

PubMed1996

The mode of action and the structure of a herbicide in complex with its target: binding of activated hydantocidin to the feedback regulation site of adenylosuccinate synthetase.

(+)-Hydantocidin, a recently discovered natural spironucleoside with potent herbicidal activity, is shown to be a proherbicide that, after phosphorylation at the 5' position, inhibits adenylosuccinate synthetase, an enzyme involved in de novo purine synthesis. The mode of binding of hydantocidin 5'-monophosphate to the target enzyme was analyzed by determining the crystal structure of the enzyme-inhibitor complex at 2.6-A resolution. It was found that adenylosuccinate synthetase binds the phosphorylated compound in the same fashion as it does adenosine 5'-monophosphate, the natural feedback regulator of this enzyme. This work provides the first crystal structure of a herbicide-target complex reported to date.

Adenylosuccinate Synthase↗

PubMed1996

Crystallization and preliminary X-ray analysis of human brain hexokinase.

Human brain hexokinase type I, expressed in Escherichia coli, has been crystallized from polyethylene glycol 8000 in the presence of inorganic phosphate. The crystals are hexagonal needles of diameter 0.25 mm, diffracting to a resolution of 3.5 A on a rotating-anode/area-detector system. The crystals belong to the space group P3(1)21/P3(2)21 with cell dimensions a = b = 171.5 A and c = 99.4 A. The specific volume of the crystal is 4.2 A3/Da, suggesting an asymmetric unit with a single 100 kDa molecule and a solvent content of 71% by volume or two molecules of hexokinase with a solvent content of 41%. The complex of hexokinase with glucose crystallizes under similar conditions, giving crystals of the same morphology.

Brain↗

PubMed1996

Refined crystal structures of guanine nucleotide complexes of adenylosuccinate synthetase from Escherichia coli.

Structures of adenylosuccinate synthetase from Escherichia coli complexed with guanosine-5'-(beta,gamma-imido) triphosphate and guanosine-5'-(beta,gamma-methylene)triphosphate in the presence and the absence of Mg2+ have been refined to R-factors below 0.2 against data to a nominal resolution of 2.7 A. Asp333 of the synthetase hydrogen bonds to the exocyclic 2-amino and endocyclic N1 groups of the guanine nucleotide base, whereas the hydroxyl of Ser414 and the backbone amide of Lys331 hydrogen bond to the 6-oxo position. The side chains of Lys331 and Pro417 pack against opposite faces of the guanine nucleotide base. The synthetase recognizes neither the N7 position of guanine nucleotides nor the ribose group. Electron density for the guanine-5'-(beta,gamma-imido) triphosphate complex is consistent with a mixture of the triphosphate nucleoside and its hydrolyzed diphosphate nucleoside bound to the active site. The base, ribose, and alpha-phosphate positions overlap, but the beta-phosphates occupy different binding sites. The binding of guanosine-5'-(beta,gamma-methylene)triphosphate to the active site is comparable with that of guanosine-5'-(beta, gamma-imido)triphosphate. No electron density, however, for the corresponding diphosphate nucleoside is observed. In addition, electron density for bound Mg2+ is absent in these nucleotide complexes. The guanine nucleotide complexes of the synthetase are compared with complexes of other GTP-binding proteins and to a preliminary structure of the complex of GDP, IMP, Mg2+, and succinate with the synthetase. The enzyme, under conditions reported here, does not undergo a conformational change in response to the binding of guanine nucleotides, and minimally IMP and/or Mg2+ must be present in order to facilitate the complete recognition of the guanine nucleotide by the synthetase.

Adenylosuccinate Synthase↗

PubMed1996

Crystallographic complexes of glucoamylase with maltooligosaccharide analogs: relationship of stereochemical distortions at the nonreducing end to the catalytic mechanism.

Crystal structures at pH 4 of complexes of glucoamylase from Aspergillus awamori var. X100 with the pseudotetrasaccharides D-gluco-dihydroacarbose and acarbose have been refined to R-factors of 0.147 and 0.131 against data to 1.7- and 2.0-A resolution, respectively. The two inhibitors bind in nearly identical manners, each exhibiting a dual binding mode with respect to the location of the last sugar residues. The reduced affinity of D-gluco-dihydroacarbose (K1 = 10(-8) M) relative to acarbose (K1 = 10(-12) M) may stem in part from the weakening of hydrogen bonds of the catalytic water (Wat 500) to the enzyme. Steric contacts between the nonreducing end of D-gluco-dihydroacarbose and the catalytic water perturb Wat 500 from its site of optimal hydrogen bonding to the active site. Interactions within the active site displace the 6-hydroxymethyl group of the nonreducing end of both acarbose and D-gluco-dihydroacarbose toward a more axial position. In the case of D-gluco-dihydroacarbose the shift in the position of the 6-hydroxymethyl group occurs with a 12 degrees change in two dihedral angles of the glucopyranose ring toward a half-chair conformation. The observed conformational distortion of the first residue of D-gluco-dihydroacarbose is consistent with the generation of a glucopyranosyl cation in the transition state. Comparable distortions of stereochemistry in model compounds require approximately 2 kcal/mol, not more than 25% of the energy necessary to form the half-chair conformation in glucose. The magnitude of stereochemical distortion observed in the active site of glucoamylase suggests that favorable electrostatic interactions between the putative glucopyranosyl cation intermediate and the active site must be more important in stabilizing the transition state than mechanical distortion of the substrate.

Acarbose↗