Kinetic studies on drug-resistant variants of Escherichia coli thymidylate synthase: functional effects of amino acid substitutions at residue 4.

A naturally occurring mutant of human thymidylate synthase (hTS) that contains a Tyr to His mutation at residue 33 was found to confer 4-fold resistance to 5-fluoro-2'-deoxyuridine (FdUrd), a prodrug of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The crystal structure of hTS implicated this Tyr residue in a drug resistance mechanistic role that may include both substrate binding and catalysis (Schiffer et al., Biochemistry, 34, 16279-16287, 1995). Because of the existence of a defined kinetic scheme and the development of a bacterial expression vector for the overproduction of Escherichia coli TS (ecTS), we chose to initially study the corresponding residue in the bacterial enzyme, Tyr 4 of ecTS. Nine mutant ecTS enzymes that differed in sequence at position 4 were generated. Mutants with a charged or polar side chain (Ser, Cys, Asp, and Arg) and Gly precipitated in the cell paste, resulting in no catalytic activity in cell-free extracts. Although most of the His 4 mutant precipitated, sufficient amounts remained in the cell-free extract to permit isolation to near homogeneity. Wild-type ecTS and mutants with a hydrophobic side chain (Phe, Ile, and Val) were expressed at nearly 30% of the total cellular protein. The k(cat) values for the isolatable mutants were 2- to 10-fold lower than that of the wild-type enzyme, while the K(m) values for 2'-deoxyuridylate (dUMP) and 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were similar for all the mutants. Dissociation constants for binary complex formation determined by stopped-flow spectroscopy were similar for the wild-type and mutant enzymes for both dUMP and 2'-deoxythymidylate, indicating that this mutation does not significantly alter the binding of the natural nucleotide ligands. However, each mutant enzyme had three- to 5-fold lower affinity for FdUMP in the binary complex compared with the wild-type enzyme, and only His 4 showed a lower affinity for FdUMP in the ternary complex. Analysis of k(burst) showed that the initial binding of CH(2)H(4)folate is weaker for each mutant compared to the wild-type enzyme and that lower k(cat) values were due to compromised rates that govern the chemical transformation of bound substrates to bound products.     

Role of Lys-226 in the catalytic mechanism of Bacillus stearothermophilus serine hydroxymethyltransferase--crystal structure and kinetic studies

Serine hydroxymethyltransferase (SHMT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme catalyzes the reversible conversion of l-Ser and tetrahydropteroylglutamate (H(4)PteGlu) to Gly and 5,10-methylene tetrahydropteroylglutamate (CH(2)-H(4)PteGlu). Biochemical and structural studies on this enzyme have implicated several residues in the catalytic mechanism, one of them being the active site lysine, which anchors PLP. It has been proposed that this residue is crucial for product expulsion. However, in other PLP-dependent enzymes, the corresponding residue has been implicated in the proton abstraction step of catalysis. In the present investigation, Lys-226 of Bacillus stearothermophilus SHMT (bsSHMT) was mutated to Met and Gln to evaluate the role of this residue in catalysis. The mutant enzymes contained 1 mol of PLP per mol of subunit suggesting that Schiff base formation with lysine is not essential for PLP binding. The 3D structure of the mutant enzymes revealed that PLP was bound at the active site in an orientation different from that of the wild-type enzyme. In the presence of substrate, the PLP ring was in an orientation superimposable with that of the external aldimine complex of wild-type enzyme. However, the mutant enzymes were inactive, and the kinetic analysis of the different steps of catalysis revealed that there was a drastic reduction in the rate of formation of the quinonoid intermediate. Analysis of these results along with the crystal structures suggested that K-226 is responsible for flipping of PLP from one orientation to another which is crucial for H(4)PteGlu-dependent Calpha-Cbeta bond cleavage of l-Ser.     

The roles of Glu-327 and His-446 in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by NMR spectroscopic and mutational analyses of the enzyme in the transient phosphohistidine intermediate complex

The bisphosphatase domain derived from the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was studied by 1H-13C HMQC NMR spectroscopy of the histidine C2' and H2' nuclei. The bacterially expressed protein was specifically labeled with 13C at the ring C2' position of the histidines. Each of the seven histidine residues gave rise to a single cross-peak in the HMQC spectra, and these were assigned by use of a series of histidine-to-alanine point mutants. His-304, His-344, and His-469 exhibit 13C and 1H resonances that titrated with pH, while the remaining histidine-associated resonances did not. The 13C and 1H chemical shifts indicate that at neutral pH, His-304 and His-446 are deprotonated, while His-469 is protonated. The pKa of His-344 was determined to be 7.04. The 13C chemical shifts suggest that the deprotonated His-258 exists as the N1' tautomer, while His-392 and His-419 are protonated in the resting, wild-type enzyme. Mutation of the remaining member of the catalytic triad, Glu-327, to alanine in the resting enzyme caused an upfield shift of 1.58 and 1.30 ppm in the 1H and 13C dimensions, respectively, and significant narrowing of the His-258 cross-peak. Mutation of His-446 to alanine produced perturbations of the His-258 cross-peak that were similar to those detected in the E327A mutant. The His-392 resonances were also shifted by the E327A and H446A mutations. These observations strongly suggest that residues His-258, Glu-327, His-392, and His-446 exist within a network of interacting residues that encompasses the catalytic site of the bisphosphatase and includes specific contacts with the C-terminal regulatory region of the enzyme. The specifically 13C-labeled bisphosphatase was monitored during turnover by HMQC spectra acquired from the transient N3' phosphohistidine intermediate complex in the wild-type enzyme, the E327A mutant, and the H446A mutant. These complexes were formed during reaction with the physiological substrate fructose-2, 6-bisphosphate. Upon formation of the phosphohistidine at His-258, the 13C and 1H resonances of this residue were shifted downfield by 1.7 and 0.31 ppm, respectively, in the wild-type enzyme. The upfield shifts of the His-258 resonances in the E327A and H446A mutant resting enzymes were reversed when the phosphohistidine was formed, generating spectra very similar to that of the wild-type enzyme in the intermediate complex. In contrast, the binding of fructose-6-phosphate, the reaction product, to the resting enzyme did not promote significant changes in the histidine-associated resonances in either the wild-type or the mutant enzymes. The interpretation of these data within the context of the X-ray crystal structures of the enzyme is used to define the role of Glu-327 in the catalytic mechanism of the bisphosphatase and to identify His-446 as a putative link in the chain of molecular events that results in activation of the bisphosphatase site by cAMP-dependent phosphorylation of the hepatic bifunctional enzyme.     

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH.     

Catalytic cysteine of thymidylate synthase is activated upon substrate binding

The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in catalysis has been characterized by kinetic and crystallographic studies. Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis. Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of E. coli in medium lacking thymidine. Steady-state kinetic analysis revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower than that of the wild-type enzyme. Relative to wild-type TS, K(m) values of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher. The rate of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was analyzed and showed that only S167A and S167G catalyzed the dehalogenation reaction and values of k(cat)/K(m) for the mutant enzymes were decreased by 10- and 3000-fold, respectively. Analysis of pre-steady-state kinetics of ternary complex formation revealed that the productive binding of CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme. Chemical transformation constants (k(chem)) for the mutant enzymes were lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to either 1.7 A (S167A and S167T) or 2.6 A (S167C). The high-resolution data sets were refined to a R(crys) of 19.9%. In the crystals some cysteine residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyethyl)thiocysteine. The pattern of derivatization indicates that in the absence of bound substrate the catalytic cysteine is not more reactive than other cysteines. It is proposed that the catalytic cysteine is activated by substrate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.     

Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism

An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The inhibition constants for arsenate and for glycerol phosphate with the mutant enzyme are similar to those with the wild-type isomerase, but the substrate analogues 2-phosphoglycolate and phosphoglycolohydroxamate bind 8- and 35-fold, respectively, more weakly to the mutant isomerase. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving 14C and 3H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. When the enzymatic reaction is conducted in tritiated solvent, the mutant isomerase does not catalyze any appreciable exchange between protons of the remaining substrate and those of the solvent either in the forward reaction direction (using dihydroxyacetone phosphate as substrate) or in the reverse direction (using glyceraldehyde phosphate as substrate). However, the specific radioactivity of the product glyceraldehyde phosphate formed in the forward reaction is 31% that of the solvent, while that of the product dihydroxyacetone phosphate formed in the reverse reaction is 24% that of the solvent. The deuterium kinetic isotope effects observed with the mutant isomerase using [1(R)-2H]dihydroxyacetone phosphate and [2-2H]glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme. The data allow us more closely to define the role of His-95 in the reaction catalyzed by the wild-type enzyme, while forcing us to be alert to subtle changes in mechanistic pathways when mutant enzymes are generated.     

Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase.

Based on predictions of the structure of proteinase 3C of poliovirus, mutations have been made at residues that are supposed to constitute the catalytic triad. Wild-type and mutant 3C were expressed in Escherichia coli, purified to homogeneity, and characterized by the ability to cleave a synthetic peptide substrate or an in vitro translated polypeptide consisting of part of the polyprotein of poliovirus. Additionally, the ability of autocatalytic processing of a precursor harboring wild-type or mutant 3C sequences was tested. Single substitutions of the residues His-40, Glu-71, and Cys-147 by Tyr, Gln, and Ser, respectively, resulted in an inactive enzyme. Replacement of Asp-85 by Asn resulted in an enzyme that was as active as wild-type enzyme in trans cleavage assays but whose autoprocessing ability was impaired. Our results are consistent with the proposal that residues His-40, Glu-71, and Cys-147 constitute the catalytic triad of poliovirus 3C proteinase. Furthermore, residue Asp-85 is not required for proper proteolytic activity despite being highly conserved between different picornaviruses. This indicates that Asp-85 might be involved in a different function of 3C.     

Assessment of amino-acid substitutions at tryptophan 16 in alpha-galactosidase.

The tryptophan residue at position 16 of coffee bean alpha-galactosidase has previously been shown to be essential for enzyme activity. The potential role of this residue in the catalytic mechanism has been further studied by using site-directed mutagenesis to substitute every other amino acid for tryptophan at that site. Mutant enzymes were expressed in Pichia pastoris, a methylotrophic yeast strain, and their kinetic parameters were calculated. Only amino acids containing aromatic rings (phenylalanine and tyrosine) were able to support a significant amount of enzyme activity, but the kinetics and pH profiles of these mutants differed from wild-type. Substitution of arginine, lysine, methionine, or cysteine at position 16 allowed a small amount of enzyme activity with the optimal pH shifted towards more acidic. All other residues abolished enzyme activity. Our data support the hypothesis that tryptophan 16 is affecting the pKa of a carboxyl group at the active site that participates in catalysis. We also describe an assay for continuously measuring enzyme kinetics using fluorogenic 4-methylumbelliferyl substrates. This is useful in screening enzymes from colonies and determining the enzyme kinetics when the enzyme concentration is not known.     

NMR analysis of site-specific mutants of yeast phosphoglycerate kinase. An investigation of the triose-binding site

Site-specific mutants of yeast phosphoglycerate kinase have been produced in order to investigate the roles of the 'basic-patch' residues, arginine 168 and histidine 170. The fully-conserved residue, arginine 168, has been replaced with a lysine (R168K) and a methionine (R168M) residue, while the non-conserved histidine 170 has been replaced with an aspartate (H170D). Comparison of the 500-MHz 1H-NMR spectra of the mutant proteins with that of wild-type phosphoglycerate kinase shows that the overall fold of the mutants remains essentially unaltered from that of the native enzyme. Results of NOE experiments indicate that there are only very minor changes in structure in the vicinity of the mutations. These mutations have also led to firm sequence-specific resonance assignments to histidines 62, 167 and 170. NMR studies of 3-phosphoglycerate binding show that decreasing the positive charge in the sequence 168-170 reduces the binding of this substrate (by about 15-fold and 4-fold for mutants R168M and H170D respectively). Mutant R168K binds 3-phosphoglycerate with an affinity about twofold less than that of the native enzyme. Significantly, the activity of mutant H170D, measured at saturating substrate concentrations, is unchanged from that of the wild-type enzyme. This indicates that this residue is not of major importance in the binding or reaction of 3-phosphoglycerate. The observation is in agreement with results obtained for the wild-type enzyme, which indicate that 3-phosphoglycerate interacts most strongly with histidine 62 and least strongly with histidine 170, as would be predicted from the X-ray crystal structure. Substitution of positively charged arginine 168 with neutral methionine (or positively charged lysine) does not cause a detectable change in the pKa values of the neighbouring histidine groups, in as much as they remain below 3. The results reported here indicate that the observed reduction in catalytic efficiency relates less to direct electrostatic effects than to the mutants' inability to undergo 3-phosphoglycerate-induced conformational changes.     

Identification of the catalytically important histidine of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

We identify His381 of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase as the basic residue functional in catalysis. The catalytic domain of 20 HMG-CoA reductases contains a single conserved histidine (His381 of the P. mevalonii enzyme). Diethyl pyrocarbonate inactivated the P. mevalonii enzyme, and hydroxylamine partially restored activity. We changed His381 to alanine, lysine, asparagine, and glutamine. The mutant proteins were overexpressed, purified to homogeneity, and characterized. His381 mutant enzymes were not inactivated by diethyl pyrocarbonate. All four mutant enzymes exhibited wild-type crystal morphology and chromatographed on substrate affinity supports like wild-type enzyme. The mutant enzymes had low catalytic activity (Vmax 0.06-0.5% that of wild-type enzyme), but Km values approximated those for wild-type enzyme. For wild-type enzyme and mutant enzymes H381A, H381N, and H381Q, Km values at pH 8.1 were 0.45, 0.27, 3.7, and 0.71 mM [(R,S)-mevalonate]; 0.05, 0.03, 0.20, and 0.11 mM [coenzyme A]; 0.22, 0.14, 0.81, and 0.62 mM [NAD+]. Km values at pH 11 for wild-type enzyme and mutant enzyme H381K were 0.32 and 0.75 mM [(R,S)-mevalonate]; 0.24 and 0.50 mM [coenzyme A]; 0.15 and 1.23 mM [NAD+]. Both pK values for the enzyme-substrate complex increased relative to wild-type enzyme (by 1-2.5 pH units for pK1 and by 0.5-1.3 pH units for pK2). For mutant enzyme H381K, the pK1 of 10.2 is consistent with lysine acting as a general base at high pH. His381 of P. mevalonii HMG-CoA reductase, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif of the catalytic domain of eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis.     

Glutamate 87 is important for menaquinol binding in DmsC of the DMSO reductase (DmsABC) from Escherichia coli

Escherichia coli dimethylsulfoxide (DMSO) reductase is a trimeric enzyme with a catalytic dimer (DmsAB) and an integral membrane anchor (DmsC). Using site-directed mutagenesis, we examined six residues in the periplasmic loop between helices two and three, potentially involved in menaquinol binding in DmsC. Mutants were characterised for growth, enzyme expression and activity, and 2-n-heptyl-4-hydroxoquinoline N-oxide (HOQNO) inhibitor binding. Mutations of leucine 66, glycine 67, arginine 71, phenylalanine 73 and serine 75 had no effect on menaquinol binding. Only a glutamate residue (E87) located in helix three was important for menaquinol binding. E87 was replaced with lysine, glutamine and aspartate. All three mutants were assembled into the membrane. Neither the lysine nor the glutamine mutant enzymes were able to support anaerobic growth on glycerol/DMSO minimal media or oxidise lapachol. The glutamine mutant bound the inhibitor with lower affinity compared to wild-type, whereas in the lysine mutant, binding was almost abolished. The aspartate mutant behaved as a wild-type enzyme. The data shows that E87 is important for menaquinol binding and oxidation and is likely to act as a proton acceptor in the menaquinol binding site.     

Cysteine-scanning mutagenesis of muscle carnitine palmitoyltransferase I reveals a single cysteine residue (Cys-305) is important for catalysis

Carnitine palmitoyltransferase (CPT) I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of l-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. To determine the role of the 15 cysteine residues in the heart/skeletal muscle isoform of CPTI (M-CPTI) on catalytic activity and malonyl-CoA sensitivity, we constructed a 6-residue N-terminal, a 9-residue C-terminal, and a 15-residue cysteineless M-CPTI by cysteine-scanning mutagenesis. Both the 9-residue C-terminal mutant enzyme and the complete 15-residue cysteineless mutant enzyme are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. Mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be important for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type, suggesting that Cys-305 is the residue involved in catalysis. Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. Based on the mutagenesis and substrate labeling studies, we propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of Cys-305, His-473, and Asp-454 with Cys-305 serving as a probable nucleophile, thus acting as a site for covalent attachment of the acyl molecule and formation of a stable acyl-enzyme intermediate. This would in turn allow carnitine to act as a second nucleophile and complete the acyl transfer reaction.     

Site-directed mutagenesis of Pro-17 located in the glycine-rich region of adenylate kinase

Proline 17 in the glycine-rich region of adenylate kinase was replaced by Gly (the Gly-mutant) or Val (the Val-mutant) by site-directed mutagenesis. The mutant enzymes were purified to homogeneous states on sodium dodecyl sulfate-gel electrophoresis after solubilization of the proteins from the pellets of cell lysates of Escherichia coli. The apparent Km values of the Gly- and the Val-mutants for AMP increased approximately 7- and 24-fold, respectively, as compared with that of the wild-type enzyme. The apparent Km values for ATP also increased 7- and 42-fold in the Gly- and Val-mutants, respectively. In contrast, Vmax values of both mutant enzymes were comparable to that of the wild-type enzyme. These results suggest that Pro-17 plays an important role for the binding of substrates, but not for catalytic efficiency, although it does not directly interact with substrates. Adenosine diphosphopyridoxal, which specifically modifies Lys-21 in adenylate kinase (Tagaya, M., Yagami, T., and Fukui, T. (1987) J. Biol. Chem. 262, 8257-8261), inactivated the wild-type and mutant enzymes at almost the same rates. Interestingly, both mutant enzymes showed higher specificities for adenine nucleotides than the wild-type enzyme. Both mutant enzymes were less resistant than the wild-type enzyme against inactivation at elevated temperatures or by treatment with trypsin. It would appear that most of the properties of the mutant enzymes may be explained on the basis of a need for conformational flexibility of the loop which includes Pro-17 for substrate binding.     

Identification of lysine 122 and arginine 196 as important functional residues of rat CTP:phosphocholine cytidylyltransferase alpha

CTP:phosphocholine cytidylyltransferase alpha (CCTalpha) contains a central region that functions as a catalytic domain, converting phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the subsequent synthesis of phosphatidylcholine. We have investigated the catalytic role of lysine 122 and arginine 196 of rat CCTalpha using site-directed mutagenesis and a baculovirus expression system. Arginine 196 is part of the highly conserved RTEGIST motif, while lysine 122 has not previously been identified by protein sequence alignment as a candidate catalytic amino acid. Removing the side chain of lysine 122 compromises the catalytic ability of CCTalpha, decreasing the apparent V(max) value in mutant enzymes Lys122Ala and Lys122Arg to 0.30 and 0.09% of the wild-type value, respectively. The decrease in V(max) is accompanied by dramatic 471- and 80-fold increases in the apparent K(m) value for phosphocholine but no greater than 3-fold increases in the apparent Hill constant (K*) value for CTP. Mutation of arginine 196 to lysine results in an enzyme that retains 24% of the wild-type V(max) value with a modest 5-fold increase in the K(m) value for phosphocholine. However, the Arg196Lys mutant enzyme exhibits a 23-fold increase in the K* value for CTP. These data suggest lysine 122 and arginine 196 of rat CTP:phosphocholine cytidylyltransferase are functionally important amino acids, perhaps at or near the active site involved in forming contacts with the substrates phosphocholine and CTP, respectively.     

Primary structure, physicochemical properties, and chemical modification of NAD(+)-dependent D-lactate dehydrogenase. Evidence for the presence of Arg-235, His-303, Tyr-101, and Trp-19 at or near the active site.

The NAD(+)-dependent D-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified D-lactate dehydrogenase gene sequence. The enzyme is a dimer of identical subunits (specific activity 2800 +/- 100 units/min at 25 degrees C). Each subunit contains 332 amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents 2,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NADH reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-235, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-235 and His-303 are involved in the binding of 2-oxo acid substrate whereas other residues are involved in binding of the cofactor.     

Comparison of ELISA with activity and ligand-binding methods for the determination of thymidylate synthase concentration.

The determination of enzyme levels in cellular extracts by active site titrations or by catalytic activity measurements is relevant in both science and medicine. However, these techniques assume that enzymes exhibit the same response in crude sample matrices as they do in the purified state. We report here an example of how an enzyme-linked immunosorbent assay (ELISA) was used to determine the true enzyme concentration which was compared to the effective enzyme concentration obtained by ligand binding and catalytic assay methods in a crude bacterial cell extract. Rabbit antibodies specific for Lactobacillus casei thymidylate synthase (TS) were used to develop a highly specific and sensitive heterogeneous noncompetitive ELISA assay with a typical detection limit of 1.4 fmol of TS (100 pg) and a dynamic working range of 3 orders of magnitude. The antibodies showed identical responses for TS, its inhibitory binary complex with 5-fluoro-2'-deoxyuridylate, and its inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate in the immunoassay. L. casei cell-free extracts were subjected to extraction with CM-Sephadex and the various fractions were analyzed by ELISA, active-site titrations, and catalytic assays which demonstrated that assays which assumed full catalytic or ligand-binding competence underestimated the true enzyme level.     

Aminoethylcysteine can replace the function of the essential active site lysine of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The biodegradative 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of Pseudomonas mevalonii catalyzes the NAD(+)-dependent conversion of (S)-HMG-CoA to (R)-mevalonate. Crystallographic analysis of abortive ternary complexes of this enzyme revealed lysine 267 located at a position in the active site, suggesting that it might serve as the general acid/base for catalysis. Site-directed mutagenesis and subsequent chemical derivatization were therefore employed to investigate this active site lysine. Replacement of lysine 267 by alanine, histidine, or arginine resulted in mutant enzymes that lacked detectable activity. Lysine 267 was next replaced by the lysine analogues aminoethylcysteine and carboxyamidomethylcysteine. Using instead of the wild-type enzyme the fully active, cysteine-free mutant enzyme C156A/C296A, lysine 267 was first replaced by cysteine. Cysteine 267 of mutant enzyme C156A/C296A/K267C was then treated with bromoethylamine or iodoacetamide to insert aminoethylcysteine (AEC) or carboxyamidomethylcysteine at position 267. The carboxyamidomethylcysteine derivative was inactive, whereas mutant enzyme C156A/C296A/K267AEC exhibited high catalytic activity. That aminoethylcysteine, but not other basic amino acids, can replace the function of lysine 267 documents both the importance of this residue and the requirement for a precisely positioned positive charge at the active site of the enzyme.     

Nonessentiality of histidine 291 of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase as determined by site-directed mutagenesis

Chemical modification of spinach ribulosebisphosphate carboxylase/oxygenase by diethyl pyrocarbonate led to the conclusion that His-298 is an essential active-site residue (Igarashi, Y., McFadden, B. A., and El-Gul, T. (1985) Biochemistry 24, 3957-3962). From the pH dependence of inactivation, the pKa of His-298 was observed to be approximately 6.8, and it was suggested that this histidine might be the essential base that initiates catalysis (Paech, C. (1985) Biochemistry 24, 3194-3199). To explore further the possible function of His-298, we have used site-directed mutagenesis to replace the corresponding residue of the Rhodospirillum rubrum carboxylase (His-291) with alanine. Assays of extracts of Escherichia coli JM107, harboring either the wild-type or mutant gene in an expression vector, revealed that the mutant protein is approximately 40% as active catalytically as the normal carboxylase. After purification to near homogeneity by immunoaffinity chromatography, the mutant protein was partially characterized with respect to subunit structure, kinetic parameters, and interaction with a transition-state analogue. The purified mutant carboxylase had a kcat of 1.5 s-1 and a kcat/Km of 1.7 X 10(4) M-1 s-1 in contrast to values of 3.6 s-1 and 6 X 10(5) M-1 s-1 for the normal enzyme. The high level of enzyme activity exhibited by the Ala-291 mutant excludes His-291 in the R. rubrum carboxylase (and by inference His-298 in the spinach carboxylase) as a catalytically essential residue.     

Functional and structural characterization of thermostable D-amino acid aminotransferases from Geobacillus spp

D-amino acid aminotransferases (D-AATs) from Geobacillus toebii SK1 and Geobacillus sp. strain KLS1 were cloned and characterized from a genetic, catalytic, and structural aspect. Although the enzymes were highly thermostable, their catalytic capability was approximately one-third of that of highly active Bacilli enzymes, with respective turnover rates of 47 and 55 s(-1) at 50 degrees C. The Geobacillus enzymes were unique and shared limited sequence identities of below 45% with D-AATs from mesophilic and thermophilic Bacillus spp., except for a hypothetical protein with a 72% identity from the G. kaustophilus genome. Structural alignments showed that most key residues were conserved in the Geobacillus enzymes, although the conservative residues just before the catalytic lysine were distinctively changed: the 140-LRcD-143 sequence in Bacillus D-AATs was 144-EYcY-147 in the Geobacillus D-AATs. When the EYcY sequence from the SK1 enzyme was mutated into LRcD, a 68% increase in catalytic activity was observed, while the binding affinity toward alpha-ketoglutarate decreased by half. The mutant was very close to the wild-type in thermal stability, indicating that the mutations did not disturb the overall structure of the enzyme. Homology modeling also suggested that the two tyrosine residues in the EYcY sequence from the Geobacillus D-AATs had a pi/pi interaction that was replaceable with the salt bridge interaction between the arginine and aspartate residues in the LRcD sequence.     

Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate.

Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP). This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis. The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme. In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH. The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme. Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme. While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate. In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme. The presteady-state reactions of these two mutant enzymes with [2-2H]aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that [kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H. (1990) Biochemistry 29, 5469-5476] for the wild-type enzyme. These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1. Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.