Catalytic role of histidine 147 in Escherichia coli thymidylate synthase

Nine mutant thymidylate synthases were isolated that only differed in sequence at position 147. The wild-type enzyme (which had a histidine residue at 147) and mutant enzymes were purified to near homogeneity and their kinetic properties were compared. Although the kcat values for the mutant enzymes were 10-10,000-fold lower than for the wild-type enzyme, the Km values for both 2'-deoxyuridylate and 5,10-methylenetetrahydrofolate were nearly identical for all the enzymes indicating that His-147 is not significantly involved in initial substrate binding. By comparing the wild-type (His-147) to the glycine (Gly-147) enzyme, the side chain of His-147 was estimated to lower the activation energy of the catalytic step by 1.6-2.9 kcal mol-1. In contrast to the wild-type enzyme, the activity of the Gly-147 enzyme decreased when the pH was raised above 7.5. The activity loss coincided with the deprotonation of a residue that had a pKa of 9.46 +/- 0.2 and an enthalpy of ionization (delta Hion) of 12.1 +/- 0.9. These values are consistent with the involvement of a lysine or an arginine residue in the catalytic process. An inspection of the rates of ternary complex formation among enzyme, 5-fluoro-2'-deoxyuridylate, and 5,10-methylenetetrahydrofolate for the mutant enzymes indicated that His-147 is not needed for the proton removal from C-5 of 2'-deoxyuridylate but rather participates in an initial catalytic step and alters the pKa value of a catalytically important lysine or arginine residue.     

Multiple replacements establish the importance of tyrosine-503 in beta-galactosidase (Escherichia coli)

Tyr-503 of beta-galactosidase was specifically replaced with Phe, His, Cys, and Lys using site-directed mutagenesis. The normal enzyme and the substituted enzymes were purified. The activities of each of the substituted enzymes with o-nitrophenyl-beta-D-galactopyranoside (ONPG) and p-nitrophenyl-beta-D-galactopyronoside (PNPG) were very low and Y503K-beta-galactosidase was essentially inactive, showing that Tyr-503 is important for activity. The stability (including tetrameric stability) of the enzymes at 4 and 25 degrees C was essentially the same as that of the wild-type enzyme and the cleavage patterns on sodium dodecyl sulfate gels after protease action were unchanged. These studies thus indicate that Tyr-503 has no noticeable influence on stability under normal conditions. The substitutions for Tyr-503 had some small effects on the binding of both substrate and inhibitor. However, both kappa 2 (glycosidic bond cleavage rate) and kappa 3 (hydrolysis rate constant) were dramatically reduced. Each substitution except that of Lys (which can be explained by electrostatic effects) gave decreases in kappa 2 and kappa 3 of roughly the same magnitude regardless of whether the substitutions were conservative or not. This strongly implies that the changes in rate were not due to conformational changes as it is very unlikely that there would be such similar decreases in the values of kappa 2 and kappa 3 for amino acids with such different structures and chemical properties if the changes in rate were due to conformational differences. The data suggest that one possible role of Tyr-503 is as a general acid/base catalyst. Profiles of the kinetic data of the enzymes as functions of pH supported the suggestion that Tyr-503 normally acts as a general acid and base catalyst. When Tyr-503 was substituted by His, a small amount of base catalytic activity seemed to be restored. The strongest evidence that Tyr-503 acts as an acid catalyst came from studies with isoquinolinium-beta-D-galactopyranoside as the substrate. The kappa cat(s) of Y503F-beta-galactosidase and of Y503C-beta-galactosidase decreased by about an order of magnitude while the rate decreases were about 3 orders of magnitude with ONPG and PNPG. The breakdown of isoquinolinium-beta-D-galactopyranoside cannot be catalyzed by acids.     

Substitution of arginine for histidine-47 in the coenzyme binding site of yeast alcohol dehydrogenase I

Molecular modeling of alcohol dehydrogenase suggests that His-47 in the yeast enzyme (His-44 in the protein sequence, corresponding to Arg-47 in the horse liver enzyme) binds the pyrophosphate of the NAD coenzyme. His-47 in the Saccharomyces cerevisiae isoenzyme I was substituted with an arginine by a directed mutation. Steady-state kinetic results at pH 7.3 and 30 degrees C of the mutant and wild-type enzymes were consistent with an ordered Bi-Bi mechanism. The substitution decreased dissociation constants by 4-fold for NAD+ and 2-fold for NADH while turnover numbers were decreased by 4-fold for ethanol oxidation and 6-fold for acetaldehyde reduction. The magnitudes of these effects are smaller than those found for the same mutation in the human liver beta enzyme, suggesting that other amino acid residues in the active site modulate the effects of the substitution. The pH dependencies of dissociation constants and other kinetic constants were similar in the two yeast enzymes. Thus, it appears that His-47 is not solely responsible for a pK value near 7 that controls activity and coenzyme binding rates in the wild-type enzyme. The small substrate deuterium isotope effect above pH 7 and the single exponential phase of NADH production during the transient oxidation of ethanol by the Arg-47 enzyme suggest that the mutation makes an isomerization of the enzyme-NAD+ complex limiting for turnover with ethanol.     

Site-directed mutagenesis of the His residues of the rat mitochondrial carnitine/acylcarnitine carrier: Implications for the role of His-29 in the transport pathway

The mitochondrial carnitine/acylcarnitine carrier (CAC) of Rattus norvegicus contains two His, His-29 and His-205. Only the first residue is conserved in all the members of the CAC subfamily and is positioned before the first of the three conserved motifs. In the homology model of CAC, His-29 is located in H1 close to the bottom of the central cavity. His-205 is the first amino acid of H5 and it is exposed towards the cytosol. The effect of substitution of the His residues on the transport function of the reconstituted mutant CACs has been analysed, in comparison with the wild-type. H29A showed very low activity, H29K and H29D were nearly inactive, whereas H205A, H205K and H205D showed activities similar to that of the wild-type. His-29 has also been substituted with Gln, Asn, Phe and Tyr. All the mutants showed very low transport function and, similarly to H29A, higher Km, reduced Vmax and altered selectivity towards (n)acylcarnitines, with the exception of H29Q, which exhibited functional properties similar to those of the wild-type. The experimental data, together with a comparative analysis of the carnitine acyltranferase active sites, indicated that His-29 forms an H-bond with the β-OH of carnitine. The substitution of His-205 led to a change of response of the CAC to the pH. The results are discussed in terms of relationships of His-29 with the molecular mechanism of translocation of the CAC.     

Determination of the roles of Glu-461 in beta-galactosidase (Escherichia coli) using site-specific mutagenesis

Site-directed substitutions (Asp, Gly, Gln, His, and Lys) were made for Glu-461 of beta-galactosidase (Escherichia coli). All substitutions resulted in loss of most activity. Substrates and a substrate analog inhibitor were bound better by the Asp-substituted enzyme than by the normal enzyme, about the same for enzyme substituted with Gly, but only poorly when Gln, His, or Lys was substituted. This shows that Glu-461 is involved in substrate binding. Binding of the positively charged transition state analog 2-aminogalactose was very much reduced with Gly, Gln, His, and Lys, whereas the Asp-substituted enzyme bound this inhibitor even better than did the wild-type enzyme. Since Asp, like Glu, is negatively charged, this strongly supports the proposal that one role of Glu-461 is to electrostatically interact with a positively charged galactosyl transition state intermediate. The substitutions also affected the ability of the enzyme to bind L-ribose, a planar analog of D-galactose that strongly inhibits beta-galactosidase activity. This indicates that the binding of a planar "galactose-like" compound is somehow mediated through Glu-461. The data indicated that the presence of Glu-461 is highly important for the acid catalytic component of kappa 2 (glycosylic bond cleavage or "galactosylation"), and therefore Glu-461 must be involved in a concerted acid catalytic reaction, presumably by stabilizing a developing carbonium ion. The kappa 2 values with o- and p-nitrophenyl-beta-D-galactopyranoside as substrates varied more or less as did the K8 values, indicating that most of the glycolytic bond breaking activity found for the enzymes from the mutants with these substrates was probably a result of strain or other such effects. The kappa 3 values (hydrolysis or "degalactosylation") of the substituted enzymes were also low, indicating that Glu-461 is important for that part of the catalysis. The enzyme with His substituted for Glu-461 had the highest kappa 3 value. This is probably a result of the formation of a covalent bond between His and the galactosyl part of the substrate.     

Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant

Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldo-keto reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 A structure of H113A mutant bound to NADP(+), and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m xylose) show that, relative to the wild type, the hydride transfer rate constant (k(7) approximately 0.16 s(-1)) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on k(cat) and k(cat)/K(m xylose) for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(k(cat)/K(m xylose)) for the wild type and H113A decreased above apparent pK(a) values of 8.85 and 7.63, respectively. The DeltapK(a) of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on k(cat) and k(cat)/K(m aldehyde) into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett sigma scale, electronic parameter coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(k(cat)) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nepsilon2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.     

Stereochemical outcome and kinetic effects of Rp- and Sp-phosphorothioate substitutions at the cleavage site of vaccinia type I DNA topoisomerase

To probe the mechanism of the reversible DNA phosphodiester bond cleavage and religation mechanism of the type I topoisomerase from vaccinia virus, we have synthesized DNA substrates carrying a single nonbridging Rp- or Sp-phosphorothioate (Ps) modification at the scissile phosphodiester (Pd) bond. Analysis of the stereochemical outcome of the net cleavage and rejoining reaction established that the reaction proceeds with retention of configuration, as expected for a double-displacement mechanism. Single-turnover kinetic studies on irreversible strand cleavage using 18/24 mer suicide substrates showed thio effects (k(Pd)/k(Ps)) of 340- and 30-fold for the Rp-Ps and Sp-Ps stereoisomers, respectively, but approximately 10-fold smaller thio effects for the reverse single-turnover religation reaction (Rp-Ps = 30 and Sp-Ps = 3). As compared to the smaller suicide cleavage substrates, approach-to-equilibrium cleavage studies using 32/32 mer substrates showed 7-9-fold smaller thio effects on cleavage, similar effects on religation, and the same ratio of the Rp to Sp thio effect as the suicide cleavage reaction ( approximately 10). In general, thio effects of 2.4-7.2-fold on the cleavage equilibrium are observed for the wild-type and H265A enzymes, suggesting differences in the interactions of the enzyme with the nonbridging sulfur in the noncovalent and covalent complexes. Studies of the cleavage, religation, and approach-to-equilibrium reactions catalyzed by the H265A active site mutant revealed a stereoselective, 11-fold decrease in the Rp-thio effect on cleavage and religation as compared to the wild-type enzyme. This result suggests that His-265 interacts with the nonbridging pro-Rp oxygen in the transition state for cleavage and religation, consistent with the arrangement of this conserved residue in the crystal structure of the human topoisomerase-DNA complex. In general, the greatest effect of thio substitution and the H265A mutation is to destabilize the transition state, with smaller effects on substrate binding. The interaction of His-265 with the pro-Rp nonbridging oxygen is inconsistent with the proposal that this conserved residue acts as a general acid in the strand cleavage reaction.     

Mutation of amino acids thought to polarize the oxaloacetate carbonyl in citrate synthase severely reduces but does not abolish activity of the enzyme

Oligonucleotide-directed mutagenesis has been used to alter two active site residues of Escherichia coli citrate synthase, histidine-305 and arginine-314. Both residues are thought to be involved in the polarization of the carbonyl group of oxaloacetate and thus facilitate attack at the carbonyl carbon by acetyl-CoA. In one mutant, designated CS305H----A, His-305 was mutated to alanine and in the other, designated CS314R----L, Arg-314 was changed to leucine. Both mutants have greatly reduced turnover numbers, less than 0.1% of the wild-type value. The dissociation constant for formation of the binary enzyme-oxaloacetate complex, Ki, OAA, is at least 950 microM for CS305H----A, and about 500 microM for CS314R----L, 28 and 15 times the wild-type value, respectively. The Michaelis constants for the two substrates, KOAA and KAcCoA, which measure the affinity of the enzyme for the catalytically significant ternary complex, are less radically altered: values of KAcCoA are actually 3.5-fold and 4.6-fold lower for CS305H----A and CS314R----L, respectively. These kinetic effects are taken to mean that both His-305 and Arg-314 are important for the successful formation of an efficient transition state, very likely by polarizing the carbonyl group of oxaloacetate as has been suggested, and that the residual kinetic activity, in both mutants, occurs by a mechanism which benefits from only part of this polarization. Allosteric properties of the mutant enzymes, as measured by NADH inhibition and binding, and KCl activation, are normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase

The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.     

Tyr-503 of beta-galactosidase (Escherichia coli) plays an important role in degalactosylation.

Substitutions for Tyr-503 of beta-galactosidase caused large decreases of the activity. Both the galactosylation (k2) and degalactosylation (k3) rates were decreased. Substitutions by residues without transferable protons, caused k3 to decrease much more than k2 while substitutions with residues having transferable protons, caused approximately equal decreases of k2 and k3. Several lines of evidence showed this. The Km values of the substituted enzymes were much smaller than those for the wild type if the substituted amino acid residues did not have transferable protons; this was not the case when the substituted residues had transferable protons. Inhibition studies showed that the Km values were not small because of small Ks values but were small because of relatively small k3 values (compared with the k2 values). The conclusion that the k3 values are small relative to k2 upon substitution with residues without transferable protons is also based upon other studies: studies indicating that the reaction rates were similar with different substrates, studies in the presence of alcohol acceptors, studies showing that the rate of inactivation by 2,4-dinitrophenyl-2-deoxy-2-F-beta-D-galactopyranoside decreased much less than the rate of reactivation; studies on burst kinetics, and pH studies. The data suggest that Tyr-503 may be important for the degalactosylation reaction because of its ability to transfer protons and thereby facilitate cleavage of the transient covalent bond between galactose and Glu-537.     

Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: mutational analysis of catalytic residues

We engineered an acetyl xylan esterase (AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser(119), Ser(146), Asp(168) and Asp(202), were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser(119) and Asp(202) are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased K(m) and decreased k(cat). The catalytic efficiency of S146A was 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold than that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.     

Beta-galactosidase (Escherichia coli) has a second catalytically important Mg2+ site

It is shown here that Escherichia coli beta-galactosidase has a second Mg2+ binding site that is important for activity. Binding of Mg2+ to the second site caused the k(cat) (with oNPG as the substrate) to increase about 100 s(-1); the Km was not affected. The Kd for binding the second Mg2+ is about 10(-4)M. Since the concentration of free Mg2+ in E. coli is about 1-2 mM, the second site is physiologically significant. Non-polar substitutions (Ala or Leu) for Glu-797, a residue in an active site loop, eliminated the k(cat) increase. This indicates that the second Mg2+ site is near to Glu-797. The Ki values of transition state analogs were decreased by small but statistically significant amounts when the second Mg2+ site was occupied and Arrhenius plots showed that less entropic activation energy is required when the second site is occupied. These inhibitor and temperature results suggest that binding of the second Mg2+ helps to order the active site for stabilization of the transition state.     

Characterization of two site-specifically mutated human dihydrolipoamide dehydrogenases (His-452----Gln and Glu-457----Gln).

Two site-specifically mutated human dihydrolipoamide dehydrogenases (His-452----Gln and Glu-457----Gln) were expressed in pyruvate dehydrogenase complex-deletion mutant Escherichia coli JRG1342. The expressed mutant E3s were purified to near homogeneity using DEAE-Sephacel and hydroxyapatite columns. The initial velocity measurements in the absence of products for the Gln-452 mutant E3 in the direction of NAD+ reduction showed parallel lines in double-reciprocal plots, indicating that the mutant E3, like wild-type enzyme, catalyzed E3 reaction via a ping-pong mechanism. The specific activity of the Gln-452 mutant E3 was about 0.2% of that of wild-type enzyme. Its Km for dihydrolipoamide was dramatically increased by 63-fold. The substitution of His-452 to Gln resulted in a destabilization of the transition state of human E3 catalysis by about 6.4 kcal mol-1. The Gln-457 mutant E3, unlike wild-type enzyme, catalyzed E3 reaction via a sequential mechanism in the direction of NAD+ reduction based on the intersecting lines shown on double-reciprocal plots. Its specific activity decreased to 28% of that of wild-type enzyme. Its Km for dihydrolipoamide increased about 4.3-fold. The substitution of Glu-457 to Gln resulted in a destabilization of the transition state by about 1.7 kcal mol-1. These results indicate that His-452, which is a possible proton acceptor/donor in human E3 reaction, is critical to human E3 catalysis and that the local environment around His-452 and Glu-457, which are suggested to be hydrogen-bonded, is important in the binding of dihydrolipoamide to the enzyme.     

Site-directed mutagenesis of 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase. Identification of residues involved in metallocenter formation and substrate binding

2,4-dichlorophenoxyacetic acid (2,4-D)/alpha-ketoglutarate (alpha-KG) dioxygenase (TfdA) is an Fe(II)-dependent enzyme that catalyzes the first step in degradation of the herbicide 2,4-D. The active site structures of a small number of enzymes within the alpha-KG-dependent dioxygenase superfamily have been characterized and shown to have a similar HXDX(50-70)HX(10)RXS arrangement of residues that make up the binding sites for Fe(II) and alpha-KG. TfdA does not have obvious homology to the dioxygenases containing the above motif but is related in sequence to eight other enzymes in the superfamily that form a distinct consensus sequence (HX(D/E)X(138-207) HX(10)R/K). Variants of TfdA were created to examine the roles of putative metal-binding residues and the functions of the other seven histidines in this protein. The H167A, H200A, H213A, H245A, and H262A forms of TfdA formed inclusion bodies when overproduced in Escherichia coli DH5alpha; however, these proteins were soluble when fused to the maltose-binding protein (MBP). MBP-TfdA exhibited kinetic parameters similar to the native enzyme. The H8A and H235A variants were catalytically similar to wild-type TfdA. MBP-H213A and H216A TfdA have elevated K(m) values for 2,4-D, and the former showed a decreased k(cat), suggesting these residues may affect substrate binding or catalysis. The H113A, D115A, MBP-H167A, MBP-H200A, MBP-H245A and MBP-H262A variants of TfdA were inactive. Gel filtration analysis revealed that the latter two proteins were highly aggregated. The remaining four inactive variants were examined in their Cu(II)-substituted forms by EPR and electron spin-echo envelope modulation (ESEEM) spectroscopic methods. Changes in EPR spectra upon addition of substrates indicated that copper was present at the active site in the H113A and D115A variants. ESEEM analysis revealed that two histidines are bound equatorially to the copper in the D115A and MBP-H167A TfdA variants. The experimental data and sequence analysis lead us to conclude that His-113, Asp-115, and His-262 are likely metal ligands in TfdA and that His-213 may aid in catalysis or binding of 2,4-D.     

Serine hydroxymethyltransferase: origin of substrate specificity.

All forms of serine hydroxymethyltransferase, for which a primary structure is known, have five threonine residues near the active-site lysyl residue (K229) that forms the internal aldimine with pyridoxal phosphate. For Escherichia coli serine hydroxymethyltransferase each of these threonine residues has been changed to an alanine residue. The resulting five mutant enzymes were purified and characterized with respect to kinetic and spectral properties. The mutant enzymes T224A and T227A showed no significant changes in kinetic and spectral properties compared to the wild-type enzyme. The T225A and T230A enzymes exhibited differences in Km and kcat values but exhibited the same spectral properties as the wild-type enzyme. The four threonine residues at positions 224, 225, 227, and 230 do not play a critical role in the mechanism of the enzyme. The T226A enzyme had nearly normal affinity for substrates and coenzymes but had only 3% of the catalytic activity of the wild-type enzyme. The spectrum of the T226A enzyme in the presence of amino acid substrates showed a large absorption maximum at 343 nm with only a small absorption band at 425 nm, unlike the wild-type enzyme whose enzyme-substrate complexes absorb at 425 nm. Rapid reaction studies showed that when amino acid substrates and substrate analogues were added to the T226A enzyme, the internal aldimine absorbing at 422 nm was rapidly converted to a complex absorbing at 343 nm in a second-order process. This was followed by a very slow first-order formation of a complex absorbing at 425 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Escherichia coli serine hydroxymethyltransferase. The role of histidine 228 in determining reaction specificity.

Serine hydroxymethyltransferase has a conserved histidine residue (His-228) next to the lysine residue (Lys-229) which forms the internal aldimine with pyridoxal 5'-phosphate. This histidine residue is also conserved at the equivalent position in all amino acid decarboxylases and tryptophan synthase. Two mutant forms of Escherichia coli serine hydroxymethyltransferase, H228N and H228D, were constructed, expressed, and purified. The properties of the wild type and mutant enzymes were studied with substrates and substrate analogs by differential scanning calorimetry, circular dichroism, steady state kinetics, and rapid reaction kinetics. The conclusions of these studies were that His-228 plays an important role in the binding and reactivity of the hydroxymethyl group of serine in the one-carbon-binding site. The mutant enzymes utilize substrates and substrate analogs more effectively for a variety of alternate non-physiological reactions compared to the wild type enzyme. As one example, the mutant enzymes cleave L-serine to glycine and formaldehyde when tetrahydropyteroylglutamate is replaced by 5-formyltetrahydropteroylglutamate. The released formaldehyde inactivates these mutant enzymes. The loss of integrity of the one-carbon-binding site with L-serine in the two mutant forms of the enzyme may be the result of these enzymes not undergoing a conformational change to a closed form of the active site when serine forms the external aldimine complex.     

Role of leucine 66 in the asymmetric recognition of substrates in chicken muscle adenylate kinase

Adenylate kinase has two distinct binding sites for nucleotide substrates, MgATP and AMP. To identify the location of the site that specifically interacts with the adenine ring of AMP, we have substituted Ala, Gly, Val, Gln, and Trp for Leu66 of the recombinant chicken muscle enzyme by site-directed mutagenesis. All the purified Leu66 mutant enzymes exhibited an essentially identical circular dichroism spectrum and had thermal stabilities similar to the wild-type enzyme. Steady state kinetic analysis showed that the Leu66 mutant enzymes have significantly decreased Vmax values and markedly large Km values only for AMP. These results show that the binding site for the adenine ring of AMP in adenylate kinase is presumably located close to Leu66, which is invariant in all the enzymes so far sequenced. Significant inhibition of activities of the mutant enzymes and quenching of the Trp66 fluorescence by substrates suggest that in some Leu66 mutant enzymes, MgATP also binds to the AMP-binding site. Thus, Leu66 of adenylate kinase might play a role in the asymmetric recognition of the adenine ring of AMP from that of MgATP. Furthermore, the hydrophobicity of the residue at position 66 appears to be important for the positive cooperativity of substrate binding.     

Tyrosine83 is essential for the activity of E. coli galactoside transacetylase

The gene (lacA) coding for Escherichia coli galactoside transacetylase was cloned into the pTrcHisB plasmid, and the corresponding hexahistidine-tagged enzyme was over-expressed and purified. The kinetic constants of the tagged protein were determined, yielding values in excellent agreement with previous observations reported for the natural enzyme. LacA Tyrosine83 was then substituted with a Valine: by comparing the K(m) and k(cat) values observed for wild type and mutant enzymes using isopropyl-thio-beta-d-galactopyranoside or p-nitrophenyl-beta-d-galactopyranoside as substrates, Tyrosine83 was identified as an essential residue for the catalytic activity of E. coli galactoside transacetylase.     

Substitution for Asn460 cripples β-galactosidase (Escherichia coli) by increasing substrate affinity and decreasing transition state stability

Substrate initially binds to β-galactosidase (Escherichia coli) at a 'shallow' site. It then moves ～3? to a 'deep' site and the transition state forms. Asn460 interacts in both sites, forming a water bridge interaction with the O3 hydroxyl of the galactosyl moiety in the shallow site and a direct H-bond with the O2 hydroxyl of the transition state in the deep site. Structural and kinetic studies were done with β-galactosidases with substitutions for Asn460. The substituted enzymes have enhanced substrate affinity in the shallow site indicating lower E·substrate complex energy levels. They have poor transition state stabilization in the deep site that is manifested by increased energy levels of the E·transition state complexes. These changes in stability result in increased activation energies and lower k(cat) values. Substrate affinity to N460D-β-galactosidase was enhanced through greater binding enthalpy (stronger H-bonds through the bridging water) while better affinity to N460T-β-galactosidase occurred because of greater binding entropy. The transition states are less stable with N460S- and N460T-β-galactosidase because of the weakening or loss of the important bond to the O2 hydroxyl of the transition state. For N460D-β-galactosidase, the transition state is less stable due to an increased entropy penalty.