Biomedical subjects

I T Weber

Publications and source records attributed to I T Weber.

At least 37 records · Page 2Linked to original sources

PubMed1997

Crystallographic analysis of human immunodeficiency virus 1 protease with an analog of the conserved CA-p2 substrate -- interactions with frequently occurring glutamic acid residue at P2' position of substrates.

Human immunodeficiency virus type 1 (HIV-1) protease hydrolysis of the Gag CA-p2 cleavage site is crucial for virion maturation and is optimal at acidic pH. To understand the processing of the CA-p2 site, we have determined the structure of HIV-1 protease complexed with an analog of the CA-p2 site, the reduced peptide inhibitor Arg-Val-Leu-r-Phe-Glu-Ala-Ahx-NH2 [r denotes the reduced peptide bond and Ahx 2-aminohexanoic acid (norleucine), respectively]. The crystal structure was refined to an R-factor of 0.17 at 0.21-nm resolution. The crystals have nearly the same lattice as related complexes in P2(1)2(1)2(1) which have twofold disordered inhibitor, but are in space group P2(1). and the asymmetric unit contains two dimers of HIV-1 protease related by 180 degrees rotation. An approximate non-crystallographic symmetry has replaced the exact crystal symmetry resulting in well-ordered inhibitor structure. Each protease dimer binds one ordered inhibitor molecule, but in opposite orientations. The interactions of the inhibitor with the two dimers are very similar for the central P2 Val to P2' Glu residues, but show more variation for the distal P3 Arg and P4' Ahx residues. Importantly, the carboxylate oxygens of Glu at P2' in the inhibitor are within hydrogen-bonding distance of a carboxylate oxygen of Asp30 of the protease suggesting that the two side chains share a proton. This interaction suggests that the enzyme-substrate complex is additionally stabilized at lower pH. The importance of this interaction is emphasized by the absence of polymorphisms of Asp30 in the protease and variants of P2' Glu in the critical CA-p2 cleavage site.

Amino Acid Sequence↗

PubMed1997

Studies on the symmetry and sequence context dependence of the HIV-1 proteinase specificity.

Two major types of cleavage sites with different sequence preferences have been proposed for the human immunodeficiency virus type 1 (HIV-1) proteinase. To understand the nature of these sequence preferences better, single and multiple amino acid substitutions were introduced into a type 1 cleavage site peptide, thus changing it to a naturally occurring type 2 cleavage site sequence. Our results indicated that the previous classification of the retroviral cleavage sites may not be generally valid and that the preference for a residue at a particular position in the substrate depends strongly on the neighboring residues, including both those at the same side and at the opposite side of the peptide backbone of the substrate. Based on these results, pseudosymmetric (palindromic) substrates were designed. The retroviral proteinases are symmetrical dimers of two identical subunits; however, the residues of naturally occurring cleavage sites do not show symmetrical arrangements, and no obvious symmetrical substrate preference has been observed for the specificity of HIV proteinase. To examine the role of the asymmetry created by the peptide bonds on the specificity of the respective primed and nonprimed halves of the binding site, amino acid substitutions were introduced into a palindromic sequence. In general, the results suggested that the asymmetry does not result in substantial differences in specificity of the S3 and S3' subsites, whereas its effect is more pronounced for the S2 and S2' subsites. Although it was possible to design several good palindromic substrates, asymmetrical arrangements may be preferred by the HIV proteinase.

HIV Protease↗

PubMed1997

Development of a novel anti-HIV-1 agent from within: effect of chimeric Vpr-containing protease cleavage site residues on virus replication.

Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag-Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection.

Anti-HIV Agents↗

PubMed1997

Activity of tethered human immunodeficiency virus 1 protease containing mutations in the flap region of one subunit.

The tethered-dimer protease of human immunodeficiency virus 1 (HIV-1) [Cheng Y.-S. E., Yin, F.H., Foundling, S., Blomstrom, D. & Kettner, C. A. (1990) Proc. Natl Acad. Sci. USA 87, 9660-9664] and its mutants containing amino acid substitutions or deletions or both in only one flap region were expressed in Escherichia coli. These mutant enzymes showed various degrees of self-processing and significantly reduced catalytic activity toward oligopeptide substrates compared with the wild type. Kinetic parameters determined for one of the oligopeptide substrates showed a dramatic increase in K(m) and decrease in Kcat values. Unexpectedly, the substrate cleavage was more efficient in low salt concentration for a mutant containing a shortened hydrophilic flap. Assays with oligopeptides representing naturally occurring cleavage sites or oligopeptides containing single amino acid substitutions at the P2 and P2' substrate positions showed only moderate changes in the substrate specificity of the mutant proteases. Predicted structures for the mutants were constructed by molecular modeling and used to interpret the results of kinetic measurements. In general, the data suggest that the mutated part of the flaps does not have a major role in determining substrate specificity; rather, it provides the hydrophobic environment and hydrogen-bond interactions with the conserved water that are necessary for efficient substrate binding and catalysis.

Amino Acid Sequence↗

PubMed1997

Analysis of comparative modeling predictions for CASP2 targets 1, 3, 9, and 17.

Comparative modeling targets 1, 3, 9 and 17 were predicted by alignment of multiple sequences and structures, when available, followed by minimization using the program AMMP. The minimization used improved potentials, and distance restraints for regions of common structure. New prediction procedures were evaluated. Three tested solvent corrections did not significantly improve the predictions. Target 17 had 85.3% sequence identity with the parent and no insertions or deletions. The prediction had a root-mean-square deviation from target 17 of 0.56 A on C alpha atoms, and 0.59 A for the ligand atoms, which verified the accuracy of the minimization. Targets 1, 3, and 9 had 36.4%, 46.7%, and 33.3% identity with the parent sequences, and predictions resulted in root-mean-square deviations for 79-85% of C alpha atoms of 1.49, 1.11, and 1.24 A, respectively. Conformational differences between parent and target crystal structures were difficult to predict. The use of distance restraints and multiple structures improved the positioning of gaps in sequence alignment. Distance restraints did not overcome errors in sequence alignment or ambiguities due to conformational variation in proteins. Predictions for targets 3 and 9 successfully reduced large deviations between parent and target structures.

Animals↗

PubMed1997

Molecular mechanics calculations on Rous sarcoma virus protease with peptide substrates.

Molecular models of Rous sarcoma virus (RSV) protease and 20 peptide substrates with single amino acid substitutions at positions from P4 to P3', where the scissile bond is between P1 and P1'. were built and compared with kinetic measurements. The unsubstituted peptide substrate. Pro-Ala-Val-Ser-Leu-Ala-Met-Thr, represents the NC-PR cleavage site of RSV protease. Models were built of two intermediates in the catalytic reaction, RSV protease with peptide substrate and with the tetrahedral intermediate. The energy minimization used an algorithm that increased the speed and eliminated a cutoff for nonbonded interactions. The calculated protease-substrate interaction energies showed correlation with the relative catalytic efficiency of peptide hydrolysis. The calculated interaction energies for the 8 RSV protease-substrate models with changes in P1 to P1' next to the scissile bond gave the highest correlation coefficient of 0.79 with the kinetic measurements, whereas all 20 substrates showed the lower, but still significant correlation of 0.46. Models of the tetrahedral reaction intermediates gave a correlation of 0.72 for the 8 substrates with changes next to the scissile bond, whereas a correlation coefficient of only 0.34 was observed for all 20 substrates. The differences between the energies calculated for the tetrahedral intermediate and the bound peptide gave the most significant correlation coefficients of 0.90 for models with changes in P1 and P1', and 0.56 for all substrates. These results are compared to those from similar calculations on HIV-1 protease and discussed in relation to the rate-limiting steps in the catalytic mechanism and the entropic contributions.

Aspartic Acid Endopeptidases↗

PubMed1997

Identification of 14 new glucokinase mutations and description of the clinical profile of 42 MODY-2 families.

Mutations in glucokinase are associated with defects in insulin secretion and hepatic glycogen synthesis resulting in mild chronic hyperglycaemia, impaired glucose tolerance or diabetes mellitus. We screened members of 35 families with features of maturity-onset diabetes of the young for mutations in the glucokinase gene and found 16 different mutations. They included 14 new mutations in the glucokinase gene: 9 missense mutations (A53S, G80A, H137R, T168P, M210T, C213R, V226M, S336L and V367M); 2 nonsense mutations (E248X and S360X); a deletion of one nucleotide resulting in a frameshift (V401del1); a substitution of a conserved nucleotide at a splice acceptor site (L122-1G-->T); and a 10 base pair deletion that removed the GT of the splice donor site and the following eight nucleotides (K161 + 2del10). In addition, we found two previously identified mutations: R186X and G261R. Study of 260 subjects with glucokinase-deficient hyperglycaemia from 42 families with 36 different GCK mutations made it possible to define the clinical profile of this subtype of non-insulin-dependent diabetes mellitus (NIDDM). Hyperglycaemia due to glucokinase deficiency is often mild (fewer than 50% of subjects have overt diabetes) and is evident during the early years of life. Despite the long duration of hyperglycaemia, glucokinase-deficient subjects have a low prevalence of micro- and macro-vascular complications of diabetes. Obesity, arterial hypertension and dyslipidaemia are also uncommon in this form of NIDDM.

Adolescent↗

PubMed1997

Crystal structure of TNF-alpha mutant R31D with greater affinity for receptor R1 compared with R2.

Crystal structures have been determined of recombinant human tumor necrosis factor-alpha (TNF-alpha) and its R31D mutant that preferentially binds to TNF receptor R1 with more than seven times the relative affinity of binding to receptor R2. Crystals of the wild-type TNF were of space group P4(1)2(1)2 and had unit cell dimensions of a = b = 94.7 and c = 117.4 A. Refinement of the structure gave an R-factor of 22.3% at 2.5 A resolution. The crystals of TNF R31D mutant diffracted to 2.3 A resolution, and were of identical space group to the wild type with unit cell dimensions of a = b = 95.4 and c = 116.2 A, and the structure was refined to an R-factor of 21.8%. The trimer structures of the wild-type and mutant TNF were similar with a root mean square (r.m.s.) deviation of 0.56 A for Calpha atoms; however, the subunits within each trimer were more variable with an average r.m.s. deviation of 1.00 A on Calpha atoms for pairwise comparison of subunits. Model complexes of TNF with receptors R1 and R2 have been used to predict TNF-receptor interactions. Arg31 in all three subunits of wild-type TNF is predicted to form an ionic interaction with the equivalent glutamic acid in both receptors R1 and R2. Asp31 of the TNF R31D mutant is predicted to interact differently with the two receptors. The side chain of Asp31 in two subunits of the TNF mutant is predicted to form hydrogen bond interactions with Ser59 or Cys70 of R1, while it has no predicted interactions with R2. The loss of three strong ionic interactions of Arg31 and the electrostatic repulsion of Asp31 with Glu in the receptors is consistent with the reduced binding of the R31D mutant to both receptors relative to wild-type TNF. The replacement of these ionic interactions by two weaker hydrogen bond interactions between Asp31 of the R31D mutant and R1, compared with no interactions with R2, is in agreement with the observed preferential binding of the R31D mutant to R1 over R2. Analysis of the structure and function of receptor-discriminating mutants of TNF will help understand the biological role of TNF and facilitate its use as an antitumor agent.

Binding Sites↗

PubMed1996

Model structure of decorin and implications for collagen fibrillogenesis.

The three-dimensional structure of human decorin, a secreted proteoglycan involved in the regulation of collagen fibrillogenesis and cellular growth, has been modeled based on the crystal structure of the porcine ribonuclease inhibitor. Both proteins contain leucine-rich repeats and share 18% identical residues. This model structure of decorin has an arch shape with the single glycosaminoglycan chain and the three N-linked oligosaccharides located on the same side of the molecule. Decorin was modeled as binding to a polar sequence of collagen type I found in the d band. The inner concave surface is the appropriate size and shape to accommodate only one collagen triple helix of approximately 3 nm in length. The binding of one collagen triple helix to decorin is proposed to play a major role in the formation of the staggered arrangement of collagen molecules within the microfibrils by preventing lateral fusion of collagen molecules.

Amino Acid Sequence↗

PubMed1996

Programming the Rous sarcoma virus protease to cleave new substrate sequences.

The Rous sarcoma virus protease displays a high degree of specificity and catalyzes the cleavage of only a limited number of amino acid sequences. This specificity is governed by interactions between side chains of eight substrate amino acids and eight corresponding subsite pockets within the homodimeric enzyme. We have examined these complex interactions in order to learn how to introduce changes into the retroviral protease (PR) that direct it to cleave substrates. Mutant enzymes with altered substrate specificity and wild-type or greater catalytic rates have been constructed previously by substituting single key amino acids in each of the eight enzyme subsites with those residues found in structurally related positions of human immunodeficiency virus (HIV)-1 PR. These individual amino acid substitutions have now been combined into one enzyme, resulting in a highly active mutant Rous sarcoma virus (RSV) protease that displays many characteristics associated with the HIV-1 enzyme. The hybrid protease is capable of catalyzing the cleavage of a set of HIV-1 viral polyprotein substrates that are not recognized by the wild-type RSV enzyme. Additionally, the modified PR is inhibited completely by the HIV-1 PR-specific inhibitor KNI-272 at concentrations where wild-type RSV PR is unaffected. These results indicate that the major determinants that dictate RSV and HIV-1 PR substrate specificity have been identified. Since the viral protease is a homodimer, the rational design of enzymes with altered specificity also requires a thorough understanding of the importance of enzyme symmetry in substrate selection. We demonstrate here that the enzyme homodimer acts symmetrically in substrate selection with each enzyme subunit being capable of recognizing both halves of a peptide substrate equally.

Aspartic Acid Endopeptidases↗

PubMed1996

Comparative studies on the substrate specificity of avian myeloblastosis virus proteinase and lentiviral proteinases.

The retroviral proteinase (PR) seems to play crucial roles in the viral life cycle, therefore it is an attractive target for chemotherapy. Previously we studied the specificity of human immunodeficiency virus (HIV) type 1 and type 2 as well as equine infectious anemia virus PRs using oligopeptide substrates. Here a similar approach is used to characterize the specificity of avian myeloblastosis virus (AMV) PR and to compare it with those of the previously characterized lentiviral PRs. All peptides representing naturally occurring Gag and Gag-Pol cleavage sites were substrates of the AMV PR. Only half of these peptides were substrates of HIV-1 PR. The Km values for AMV PR were in a micromolar range previously found for the lentiviral PRs; however, the kcat values were in a 10 30-fold lower range. A series of peptides containing single amino acid substitutions in a sequence representing a naturally occurring HIV cleavage site was used to characterize the seven substrate binding subsites of the AMV PR. The largest differences were found at the P4 and P2 positions of the substrate. Detailed analysis of the results by molecular modeling and comparison with previously reported data revealed the common characteristics of the specificity of the retroviral PRs as well as its strong dependence on the sequence context of the substrate.

Amino Acid Sequence↗

PubMed1996

Human immunodeficiency virus, type 1 protease substrate specificity is limited by interactions between substrate amino acids bound in adjacent enzyme subsites.

The specificity of the retroviral protease is determined by the ability of substrate amino acid side chains to bind into eight individual subsites within the enzyme. Although the subsites are able to act somewhat independently in selection of amino acid side chains that fit into each pocket, significant interactions exist between individual subsites that substantially limit the number of cleavable amino acid sequences. The substrate peptide binds within the enzyme in an extended anti-parallel beta sheet conformation with substrate amino acid side chains adjacent in the linear sequence extending in opposite directions in the enzyme-substrate complex. From this geometry, we have defined both cis and trans steric interactions, which have been characterized by a steady state kinetic analysis of human immunodeficiency virus, type-1 protease using a series of peptide substrates that are derivatives of the avian leukosis/sarcoma virus nucleocapsid-protease cleavage site. These peptides contain both single and double amino acid substitutions in seven positions of the minimum length substrate required by the retroviral protease for specific and efficient cleavage. Steady state kinetic data from the single amino acid substituted peptides were used to predict effects on protease-catalyzed cleavage of corresponding double substituted peptide substrates. The calculated Gibbs' free energy changes were compared with actual experimental values in order to determine how the fit of a substrate amino acid in one subsite influences the fit of amino acids in adjacent subsites. Analysis of these data shows that substrate specificity is limited by steric interactions between pairs of enzyme subsites. Moreover, certain enzyme subsites are relatively tolerant of substitutions in the substrate and exert little effect on adjacent subsites, whereas others are more restrictive and have marked influence on adjacent cis and trans subsites.

Amino Acid Sequence↗

PubMed1996

Predicted ligand interactions of 3'5'-cyclic nucleotide-gated channel binding sites: comparison of retina and olfactory binding site models.

Cyclic nucleotide-gated channels (CNGC) open in response to the binding of 3'5'-cyclic nucleotides. Members of the CNGC family vary as much as 100-fold in their ability to respond to cAMP and cGMP. Molecular models of the nucleotide binding domains of the bovine retina and catfish and rat olfactory CNGCs were built from the crystal structure of cAMP bound to catabolite gene activator protein (CAP) with AMMP, a program for molecular mechanics and dynamics. The nucleotide conformation can be predicted from the number of strong and weak interactions between the purine ring and the binding site. The amino acids predicted to be important for determining the nucleotide affinity and specificity are residues 61, 83 (mediated through a water molecule), 119 and 127 (CAP sequence numbers) which interact with the purine ring. These residues also dictate the conformation of the ligand in the binding pocket. cGMP is preferentially bound in the syn conformation in bovine retina, bovine olfactory and rat olfactory CNGCs due to Thr83, while either conformation can bind in catfish olfactory CNGC. cAMP is predicted to bind either in syn or anti conformation, depending on the interaction with residue 119: the anti conformation is preferentially bound in olfactory CNGCs.

Animals↗

PubMed1996

Molecular mechanics calculations on HIV-1 protease with peptide substrates correlate with experimental data.

Molecular models of HIV-1 protease and 21 peptide substrates with single amino acid substitutions at positions from P4 to P3' were built and compared with kinetic measurements. The crystal structure of HIV-1 protease with a peptidic inhibitor was modified to model the peptide substrate Pro-Ala-Val-Ser-Leu-Ala-Met-Thr for the starting geometry. Models were built of two reaction intermediates, HIV protease with peptide substrate and with its tetrahedral intermediate. The energy minimization used a new algorithm that increased the speed and eliminated a cut-off for non-bonded interactions. After minimization the models for substrate and tetrahedral intermediate both had root mean square deviations of 0.48 A for all atoms of the HIV protease compared to the starting crystal structure. Differences in the model structures and interaction energies for HIV protease with different substrates were analyzed. The calculated interaction energies for the 21 HIV protease-tetrahedral intermediate models gave a correlation coefficient of 0.64 with the kinetic measurements. The eight substrates with changes in the P1 and P1' residues next to the scissile bond gave the highest correlation of 0.93, while the 14 substrates with changes in P2-P2' gave a correlation coefficient of 0.86. The catalytic mechanism and factors influencing the catalytic efficiency of the different substrates are discussed in relation to the models. The predictive ability of molecular mechanics calculations is discussed in the context of the statistical mechanics analysis of the differences in free energy.

Crystallography, X-Ray↗

PubMed1996

Glucokinase mutations, insulin secretion, and diabetes mellitus.

The glycolytic enzyme glucokinase plays a key role in glucose sensing by the insulin-secreting pancreatic beta-cells, and mutations in the gene encoding this enzyme are a common cause of maturity-onset diabetes of the young (MODY), a form of non-insulin-dependent diabetes mellitus characterized by autosomal-dominant inheritance and onset before 25 years of age. Twenty-eight different mutations in this gene have been identified in subjects with MODY. Clinical studies have shown that subjects with MODY due to mutations in glucokinase have elevated fasting and postprandial glucose levels with normal first-phase insulin secretory responses to intravenous glucose injection and normal insulin secretion rates over a 24-h period. However, the dose-response curve relating glucose and insulin secretion rate obtained during graded intravenous glucose infusions was shifted to the right in subjects with glucokinase mutations, indicating decreased sensitivity to glucose. In normal subjects, the beta-cell was most sensitive to an increase in glucose concentration between 5.5 and 6.0 mM, whereas in patients with glucokinase mutations, the maximum responsiveness was increased to 6.5 to 7.5 mM glucose. These studies indicate that glucokinase is an important component of the glucose-sensing mechanism of the beta-cell.

Animals↗

PubMed1995

Mutational analysis of the substrate binding pocket of murine leukemia virus protease and comparison with human immunodeficiency virus proteases.

The differences in substrate specificity between Moloney murine leukemia virus protease (MuLV PR) and human immunodeficiency virus (HIV) PR were investigated by site-directed mutagenesis. Various amino acids, which are predicted to form the substrate binding site of MuLV PR, were replaced by the equivalent ones in HIV-1 and HIV-2 PRs. The expressed mutants were assayed with the substrate Val-Ser-Gln-Asn-Tyr decreases Pro-Ile-Val-Gln-NH2 (decreases indicates the cleavage site) and a series of analogs containing single amino acid substitutions in positions P4(Ser) to P3'(Val). Mutations at the predicted S2/S2' subsites of MuLV PR have a strong influence on the substrate specificity of this enzyme, as observed with mutants H37D, V39I, V54I, A57I, and L92I. On the other hand, substitutions at the flap region of MuLV PR often rendered enzymes with low activity (e.g. W53I/Q55G). Three amino acids (His-37, Val-39, and Ala-57) were identified as the major determinants of the differences in substrate specificity between MuLV and HIV PRs.

Amino Acid Sequence↗

PubMed1995

Sugar specificity of human beta-cell glucokinase: correlation of molecular models with kinetic measurements.

Human beta-cell glucokinase recognition and phosphorylation of different sugars was investigated by steady-state kinetic analysis, measurements of substrate-induced intrinsic fluorescence changes, and molecular modeling and calculation of interaction energies. Measurements of kcat/Km showed that glucokinase phosphorylated the sugars in the order glucose = mannose > deoxyglucose > fructose = glucosamine. The mode of binding of these sugars to the open conformation of glucokinase was predicted from molecular modeling. Glucokinase is predicted to form similar interactions with the 6-OH, 4-OH, and 1-OH groups of all these sugars. The interactions of the 2-OH and 3-OH groups differ and depend on the type of sugar and reflect differences in cooperative behavior. For example, glucose and deoxyglucose exhibited cooperative behavior with Hill coefficients of 1.8 and 1.5, respectively, while mannose and fructose demonstrated Michaelis-Menten behavior. Galactose, allose, and 2,5-anhydroglucitol were not substrates under the assay conditions used, and the alpha- and beta-anomers of methylglucose were poor substrates with Km's greater than 1000 mM. Glucokinase exhibited an ATPase activity which was 1/2000th that of the rate of the kinase reaction, and unlike yeast hexokinase, it was not affected by the addition of lyxose. Glucosamine was a low affinity inhibitor as well as a substrate, while N-acetylglucosamine and mannoheptulose were high-affinity inhibitors. The change in intrinsic fluorescence that was induced by glucose, mannose, and mannoheptulose had the opposite sign for glucosamine, which implies a very different mode of binding from the other sugars. The calculated interaction energies of glucokinase with glucose, mannose, deoxyglucose, and fructose agree very well with the measured values of kcat/Km, which indicates that these sugars are recognized by binding to the open conformation of glucokinase.

Adenosine Triphosphate↗

PubMed1995

Human beta-cell glucokinase. Dual role of Ser-151 in catalysis and hexose affinity.

Glucokinase is distinguished from yeast hexokinase and low Km mammalian hexokinases by its low affinity for glucose and its cooperative behavior, even though glucose binding residues and catalytic residues are highly conserved in all of these forms of hexokinase. The roles of Ser-151 and Asn-166 as determinants of hexose affinity and cooperative behavior of human glucokinase have been evaluated by site-directed mutagenesis, expression and purification of the wild-type and mutant enzymes, and steady-state kinetic analysis. Mutation of Asn-166 to arginine increased apparent affinity for both glucose and ATP by a factor of 3. Mutation of Ser-151 to cysteine, alanine, or glycine lowered the Km for glucose by factors of 2-, 26-, and 40-fold, respectively, decreased Vmax, abolished cooperativity for glucose, and also decreased Km for mannose and fructose. The Ser-151 mutants had hexose Km values similar to those of yeast hexokinase, hexokinase I, and the recombinantly expressed COOH-terminal half of hexokinase I. However, the Ki values for the competitive inhibitors, N-acetylglucosamine and glucose-6-P, were unchanged, suggesting that Ser-151 is not important for inhibitor binding. Mutation of Ser-151 also increased the Km for ATP about 5-fold and abolished the enzyme's low ATPase activity, which indicates it is essential for ATP hydrolysis. The substrate-induced change in intrinsic fluorescence of S151A occurred at a much lower glucose concentration than that for wild-type enzyme. The results implicate a dual role for Ser-151 as a determinant of hexose affinity and catalysis, exclusive of the glucose-induced conformational change, and suggest that the low hexose affinity of glucokinase is dependent on interaction of Ser-151 with other regions of the protein.

Acetylglucosamine↗