Oligonucleotide site directed mutagenesis of all histidine residues within the T4 endonuclease V gene: role in enzyme-nontarget DNA binding

In order to evaluate the contributions that histidine residues might play both in the catalytic activities of endonuclease V and in binding to nontarget DNA, the technique of oligonucleotide site directed mutagenesis was used to create mutations at each of the four histidine residues in the endonuclease V gene. Although none of the histidines were shown to be absolutely required for the pyrimidine dimer specific DNA glycosylase activity or the apurinic lyase activity, conservative amino acid changes at His16 produced enzymes with little or no catalytic activity. In addition, the evaluation of conservative and radical amino acid substitutions at positions 34, 56, and 107 is consistent with the interpretation that each of these histidines may be involved in nontarget DNA binding. The data supporting this conclusion are that histidine changes to lysine at positions 34 and 107 enhance the nontarget DNA binding activity of the mutant enzymes while neutralization of charge at His56 reduces nontarget DNA binding.     

Mutational analysis reveals only one catalytic histidine in neutral endopeptidase ("enkephalinase").

Aside from serving as zinc ligands, kinetic data has implicated one or more additional histidines as catalytic residues in neutral endopeptidase ("enkephalinase") action. One of these histidines has previously been identified as histidine 704 (Bateman et al., J. Biol. Chem., 265:8365-8368, 1990). In order to determine whether a second histidine is involved in catalysis each of these residues not previously changed have been converted to glutamine by site directed mutagenesis. The resultant recombinant enzymes possess full catalytic activity indicating that histidine 704 is the only catalytic histidine in the enzyme.     

Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase.

The conversion of thyroxine to 3,5,3'-triiodothyronine (T3) is the first step in thyroid hormone action, and the Type I iodothyronine deiodinase supplies most of this extrathyroidal T3 in the rat. We found that the cDNA coding for this enzyme contains an in-frame UGA encoding the rare amino acid selenocysteine. Using site-directed mutagenesis, we have converted selenocysteine to cysteine and expressed the wild-type and cysteine mutant enzymes in JEG-3 cells by transient transfection. The kinetic properties of the transiently expressed wild-type enzyme are nearly identical to those reported for rat liver Type I deiodinase. Substitution of sulfur for selenium causes a 10-fold increase in the Km of the enzyme for the favored substrate 3,3',5'-triiodothyronine (rT3), a 100-fold decrease in the sensitivity of rT3 deiodination to competitive inhibition by gold and a 300-fold increase in the apparent Ki for uncompetitive inhibition by 6-n-propylthiouracil. These results demonstrate that selenium is responsible for the biochemical properties which characterize Type I iodothyronine monodeiodination.     

Mutagenesis studies on the iron binding ligands of clavaminic acid synthase

Mutagenesis studies on conserved histidine residues identified as possible metal binding ligands in clavaminic acid synthase isozyme 2 were consistent with His-145 and His-280 acting as iron ligands, in support of crystallographic and previous mutagenesis studies. Mutagenesis of the four cysteines and a glutamine residue, conserved in both clavaminic acid synthase isozymes 1 and 2, demonstrated that none of these residues is essential for activity.     

Mutation analysis of the histidine residues in the glycylglycine endopeptidase ALE-1

A novel staphylolytic enzyme, ALE-1, is a glycylglycine endopeptidase produced by Staphylococcus capitis EPK1. ALE-1 possesses seven histidines. Chemical modification studies using diethylpyrocarbonate and iodoacetic acid suggested that a histidine or tyrosine residue(s) in the molecule is important for the organism's staphylolytic activity. All of the histidine residues, one tyrosine, and one aspartic acid residue in the N-terminally truncated ALE-1 (DeltaN-term ALE-1) were systematically altered by site-directed mutagenesis, and the enzyme activities and metal contents of the variants were measured. Our studies indicated that His-150, His-200, His-231, His-233, and Asp-154 are essential for the enzyme activity of DeltaN-term ALE-1. Except for His-150 and Asp-154, all of these amino acids were located within the 38-amino-acid region conserved among 11 proteins, including 5 staphylolytic endopeptidases. Inductively coupled plasma-mass spectrometric analysis of DeltaN-term ALE-1 revealed that it contains one atom of zinc per molecule. Measurement of the zinc content of the mutant DeltaN-term ALE-1 suggested that His-150 and -233 are important for zinc binding; their loss in these variant enzymes coincided with the loss of staphylolytic activity. These results strongly suggest that ALE-1 is a novel member of zinc metalloproteases.     

Histidine 295 and histidine 510 are crucial for the enzymatic degradation of heparan sulfate by heparinase III

The heparinases from Flavobacterium heparinum are powerful tools in understanding how heparin-like glycosaminoglycans function biologically. Heparinase III is the unique member of the heparinase family of heparin-degrading lyases that recognizes the ubiquitous cell-surface heparan sulfate proteoglycans as its primary substrate. Given that both heparinase I and heparinase II contain catalytically critical histidines, we examined the role of histidine in heparinase III. Through a series of diethyl pyrocarbonate modification experiments, it was found that surface-exposed histidines are modified in a concentration-dependent fashion and that this modification results in inactivation of the enzyme (k(inact) = 0.20 +/- 0.04 min(-)(1) mM(-)(1)). The DEPC modification was pH dependent and reversible by hydroxylamine, indicating that histidines are the sole residue being modified. As previously observed for heparinases I and II, substrate protection experiments slowed the inactivation kinetics, suggesting that the modified residue(s) was (were) in or proximal to the active site of the enzyme. Proteolytic mapping experiments, taken together with site-directed mutagenesis studies, confirm the chemical modification experiments and point to two histidines, histidine 295 and histidine 510, as being essential for heparinase III enzymatic activity.     

On the role of conserved histidine 106 in 10-formyltetrahydrofolate dehydrogenase catalysis: connection between hydrolase and dehydrogenase mechanisms

The enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate in an NADP(+)-dependent dehydrogenase reaction or an NADP(+)-independent hydrolase reaction. The hydrolase reaction occurs in a 310-amino acid long amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. The amino-terminal domain of FDH shares some sequence identity with several other enzymes utilizing 10-formyl-THF as a substrate. These enzymes have two strictly conserved residues, aspartate and histidine, in the putative catalytic center. We have shown recently that the conserved aspartate is involved in FDH catalysis. In the present work we studied the role of the conserved histidine, His(106), in FDH function. Site-directed mutagenesis experiments showed that replacement of the histidine with alanine, asparagine, aspartate, glutamate, glutamine, or arginine in N(t)-FDH resulted in expression of insoluble proteins. Replacement of the histidine with another positively charged residue, lysine, produced a soluble mutant with no hydrolase activity. The insoluble mutants refolded from inclusion bodies adopted a conformation inherent to the wild-type N(t)-FDH, but they did not exhibit any hydrolase activity. Substitution of alanine for three non-conserved histidines located close to the conserved one did not reveal any significant changes in the hydrolase activity of N(t)-FDH. Expressed full-length FDH with the substitution of lysine for the His(106) completely lost both the hydrolase and dehydrogenase activities. Thus, our study showed that His(106), besides being an important structural residue, is also directly involved in both the hydrolase and dehydrogenase mechanisms of FDH. Modeling of the putative hydrolase catalytic center/folate-binding site suggested that the catalytic residues, aspartate and histidine, are unlikely to be adjacent to the catalytic cysteine in the aldehyde dehydrogenase catalytic center. We hypothesize that 10-formyl-THF dehydrogenase reaction is not an independent reaction but is a combination of hydrolase and aldehyde dehydrogenase reactions.     

Mechanism of chorismate synthase. Role of the two invariant histidine residues in the active site

Chorismate synthase catalyzes the last step in the common shikimate pathway leading to aromatic compounds such as the aromatic amino acids. The reaction consists of the 1,4-anti-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate. Although this reaction does not involve a net redox change, the enzyme has an absolute requirement for reduced flavin mononucleotide, which is not consumed during the reaction. Two invariant histidine residues are found in the active site of the enzyme: His(17) and His(106). Using site-directed mutagenesis, both histidines were replaced by alanine, reducing the activity 10- and 20-fold in the H106A and H17A mutant protein, respectively. Based on the characterization of the two single mutant proteins, it is proposed that His(106) serves to protonate the monoanionic reduced FMN, whereas His(17) protonates the leaving phosphate group of the substrate. An enzymatic reaction mechanism in keeping with the experimental results is presented.     

Ethoxyformylation of bovine growth hormone

Reactivity of histidines in bovine growth hormone towards ethoxyformic anhydride was investigated and localization in the molecule of two kinetically distinguishable classes was achieved, a slow class including only histidine residue 169 (k = 0.180 min-1) and a fast one composed of histidines 19 and 21 (k = 0.900 min-1). Total ethoxyformylation of bovine growth hormone brought about a complete loss of its capacity to compete with 125I-labelled hormone for rat-liver binding sites, but modification of approximately half of the fast histidine group was enough to produce an important decrease in this capacity. Circular dichroism studies indicated no significant changes in protein conformation with all three histidine residues modified. Practically full binding capacity was restored when these residues were regenerated by treatment with hydroxylamine. These results suggest that one or both of the fast reacting histidine residues are involved in bovine growth hormone binding to its specific receptors.     

Nitrate reductase of Neurospora crassa: the functional role of individual amino acids in the heme domain as examined by site-directed mutagenesis

The enzyme nitrate reductase, which catalyzes the reduction of nitrate to nitrite, is a multi-redox center homodimeric protein. Each polypeptide subunit is approximately 100 kDa in size and contains three separate domains, one each for a flavin, a heme-iron, and a molybdopterin cofactor. The heme-iron domain of nitrate reductase has homology with the simple redox protein, cytochrome b5, whose crystal structure was used to predict a three-dimensional structure for the heme domain. Two histidine residues have been identified that appear to coordinate the iron of the heme moiety, while other residues may be important in the folding or the function of the heme pocket. Site-directed mutagenesis was employed to obtain mutants that encode nitrate reductase derivatives with eight different single amino acid substitutions within the heme domain, including the two central histidine residues. Replacement of one of these histidines by alanine resulted in a completely nonfunctional enzyme whereas replacement of the other histidine resulted in a stable and functional enzyme with a lower affinity for heme. Certain amino acid substitutions appeared to cause a rapid turnover of the heme domain, whereas other substitutions were tolerated and yielded a stable and fully active enzyme. Three different single amino acid replacements within the heme domain led to a dramatic change in regulation of nitrate reductase synthesis, with significant expression of the enzyme even in the absence of nitrate induction.     

The presence of essential histidine residues in manganese(III)-containing acid phosphatase from sweet potato

Chemical modification studies of manganese(III)-containing acid phosphatase [EC 3.1.3.2] were carried out to investigate the contributions of specific amino-acid side-chains to the catalytic activity. Incubation of the enzyme with N-ethylmaleimide at pH 7.0 caused a significant loss of the enzyme activity. The inactivation followed pseudo-first-order kinetics. Double log plots of pseudo-first-order rate constant vs. concentration gave a straight line with a slope of 1.02, suggesting that the reaction of one molecule of reagent per active site is associated with activity loss. The enzyme was protected from inactivation by the presence of molybdate or phosphate ions. Amino acid analyses of the N-ethylmaleimide-modified enzyme showed that the 96%-inactivated enzyme had lost about one histidine and one-half lysine residue per enzyme subunit without any significant decrease in other amino acids, and also demonstrated that loss of catalytic activity occurred in parallel with the loss of histidine residue rather than that of lysine residue. Molybdate ions also protected the enzyme against modification of the histidine residue. The enzyme was inactivated by photooxidation mediated by methylene blue according to pseudo-first-order kinetics. The pH profile of the inactivation rates of the enzyme showed that an amino acid residue having a pKa value of approximately 7.2 was involved in the inactivation. These studies indicate that at least one histidine residue per enzyme subunit participates in the catalytic function of Mn(III)-acid phosphatase.     

Studies on the reaction mechanism of a microbial lipase/acyltransferase using chemical modification and site-directed mutagenesis

Aeromonas hydrophila releases a protein which is a member of the lipase superfamily, similar in reaction mechanism to the important mammalian plasma enzyme lecithin-cholesterol acyltransferase. We have used chemical modification and site-directed mutagenesis of the protein to identify amino acids which may be involved in catalysis. The enzyme was unaffected by phenylmethylsulfonyl fluoride, but it was almost completely inhibited by another serine-reactive compound, diethyl p-nitrophenyl phosphate. A serine selectively modified by this reagent was identified by sequencing the amino-terminal region of the protein. It was located at position 16, in the short consensus sequence shared by the enzyme with other lipases. When this serine was changed to asparagine the product was an enzymatically inert protein which nevertheless retained the surface activity of the wild-type enzyme, suggesting its ability to bind to substrate was not changed. Diethylpyrocarbonate treatment drastically reduced the rate of acyl transfer by the native enzyme, but this did not appear to be due to modification of an essential histidine, since inhibition was not reversed by addition of hydroxylamine. We have shown that only two of the histidines in the enzyme can be involved in catalysis (Hilton, S., McCubbin, W. D., Kay, C.M., and Buckley, J. T. (1990) Biochemistry, 29, 9072-9078). Replacing both of these with asparagines had little or no effect on enzyme activity. These results indicate that, in apparent contrast to other lipases, histidine does not participate in the reaction catalyzed by the microbial enzyme. Since catalysis was not inhibited by sulfhydryl reagents, we conclude that a free cysteine is also not required for activity. This may distinguish the microbial enzyme from the mammalian acyltransferase.     

Nitrate reductase of Neurospora crassa: the functional role of individual amino acids in the heme domain as examined by site-directed mutagenesis

The enzyme nitrate reductase, which catalyzes the reduction of nitrate to nitrite, is a multi-redox center homodimeric protein. Each polypeptide subunit is approximately 100 kDa in size and contains three separate domains, one each for a flavin, a heme-iron, and a molybdopterin cofactor. The heme-iron domain of nitrate reductase has homology with the simple redox protein, cytochrome b5, whose crystal structure was used to predict a three-dimensional structure for the heme domain. Two histidine residues have been identified that appear to coordinate the iron of the heme moiety, while other residues may be important in the folding or the function of the heme pocket. Site-directed mutagenesis was employed to obtain mutants that encode nitrate reductase derivatives with eight different single amino acid substitutions within the heme domain, including the two central histidine residues. Replacement of one of these histidines by alanine resulted in a completely nonfunctional enzyme whereas replacement of the other histidine resulted in a stable and functional enzyme with a lower affinity for heme. Certain amino acid substitutions appeared to cause a rapid turnover of the heme domain, whereas other substitutions were tolerated and yielded a stable and fully active enzyme. Three different single amino acid replacements within the heme domain led to a dramatic change in regulation of nitrate reductase synthesis, with significant expression of the enzyme even in the absence of nitrate induction.     

Identification of essential residues in 2',3'-cyclic nucleotide 3'-phosphodiesterase. Chemical modification and site-directed mutagenesis to investigate the role of cysteine and histidine residues in enzymatic activity

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP; EC ) catalyzes in vitro hydrolysis of 3'-phosphodiester bonds in 2',3'-cyclic nucleotides to produce 2'-nucleotides exclusively. N-terminal deletion mapping of the C-terminal two-thirds of recombinant rat CNP1 identified a region that possesses the catalytic domain, with further truncations abolishing activity. Proteolysis and kinetic analysis indicated that this domain forms a compact globular structure and contains all of the catalytically essential features. Subsequently, this catalytic fragment of CNP1 (CNP-CF) was used for chemical modification studies to identify amino acid residues essential for activity. 5,5'-Dithiobis-(2-nitrobenzoic acid) modification studies and kinetic analysis of cysteine CNP-CF mutants revealed the nonessential role of cysteines for enzymatic activity. On the other hand, modification studies with diethyl pyrocarbonate indicated that two histidines are essential for CNPase activity. Consequently, the only two conserved histidines, His-230 and His-309, were mutated to phenylalanine and leucine. All four histidine mutants had k(cat) values 1000-fold lower than wild-type CNP-CF, but K(m) values were similar. Circular dichroism studies demonstrated that the low catalytic activities of the histidine mutants were not due to gross changes in secondary structure. Taken together, these results demonstrate that both histidines assume critical roles for catalysis.     

Site-directed mutagenesis of glutathione S-transferase YaYa: nonessential role of histidine in catalysis

A cDNA encoding a rat liver glutathione S-transferase Ya subunit has been expressed in Escherichia coli and the expressed enzyme purified to homogeneity. In order to examine the catalytic role of histidine in the glutathione S-transferase Ya homodimer, site-directed mutagenesis was used to replace all three histidine residues (at positions 8, 143, and 159) by other amino acid residues. The replacement of histidine 8 or histidine 143 with valine did not affect the 1-chloro-2,4-dinitrobenzene-conjugating activity nor the isomerase activity. However, the replacement of histidine with valine at position 159 produced the mutant GST which exhibited only partial activity. A greater decrease in catalytic activity was observed by histidine----tyrosine or histidine----lysine replacement at position 159. On the other hand, the histidine 159----asparagine mutant retained full catalytic activity. Our results indicate that histidine residues in the Ya homodimer are not essential for catalytic activity. However, histidine 159 might be critical in maintaining the proper conformation of this enzyme since replacement of this amino acid by either lysine or tyrosine did result in significant loss of enzymatic activity.     

The role of histidines in the acetate kinase from Methanosarcina thermophila

The role of histidine in the catalytic mechanism of acetate kinase from Methanosarcina thermophila was investigated by diethylpyrocarbonate inactivation and site-directed mutagenesis. Inactivation was accompanied by an increase in absorbance at 240 nm with no change in absorbance at 280 nm, and treatment of the inactivated enzyme with hydroxylamine restored 95% activity, results that indicated diethylpyrocarbonate inactivates the enzyme by the specific modification of histidine. The substrates ATP, ADP, acetate, and acetyl phosphate protected against inactivation suggesting at least one active site where histidine is modified. Correlation of residual activity with the number of histidines modified, as determined by absorbance at 240 nm, indicated that a maximum of three histidines are modified per subunit, two of which are essential for full inactivation. Comparison of the M. thermophila acetate kinase sequence with 56 putative acetate kinase sequences revealed eight highly conserved histidines, three of which (His-123, His-180, and His-208) are perfectly conserved. Diethylpyrocarbonate inactivation of the eight histidine --> alanine variants indicated that His-180 and His-123 are in the active site and that the modification of both is necessary for full inactivation. Kinetic analyses of the eight variants showed that no other histidines are important for activity. Analysis of additional His-180 variants indicated that phosphorylation of His-180 is not essential for catalysis. Possible functions of His-180 are discussed.     

Purification and characterization of ascorbic acid 2-kinase from Flavobacterium devorans ATCC 10829

Maximum activity for phosphorylating C(2)-OH of the ascorbic acid was observed at the time of 16 h incubation from the culture of Flavobacterium devorans ATCC 10829. The enzyme was purified 1.178-fold, via ammonium sulfate fractionation, Fast Q anion exchange, and phenyl agarose chromatography. Gel chromatography and SDS-polyacrylamide electrophoresis experiments showed that the enzyme is a tetramer with subunit MW of 29 kDa. Among available second substrates, pyrophosphate showed the highest activity. Optimum temperature and pH were 45 degrees C and 5.5, respectively. The enzyme was chemically modified only by diethylpyrocarbonate and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), indicating that histidine and carboxylate are in the active site. pH studies showed that two histidines are involved in the binding of the substrates and a carboxylate in catalysis. Therefore, the chemical mechanism of the enzyme is likely that two histidines bind to pyrophosphate and carboxylate, respectively, and a carboxylate acts as a general base.     

The role of histidine residues of formate dehydrogenase from Bacterium sp. 1]

The reaction between formate dehydrogenase from Bacterium sp. 1 and diethylpyrocarbonate results in the enzyme inactivation. 4 histidine residues can be blocked per subunit by this reagent. The enzyme activity correlates with the disappearance of free histidines. The process of enzyme inactivation is biphasic and obeys pseudo-first-order kinetics. NAD and NADH slow down the rate of inactivation, but do not protect histidine residues against modification. Formate does not protect the enzyme. The modification of 80% of histidines increases the Km value for both substrates 3-fold. The general conformation of enzyme in the course of modification is preserved. The modification of histidines markedly decreases the reactivity of an essential SH-group of formate dehydrogenase against the Ellman reagent.     

Functional groups in the activity and regulation of Escherichia coli citrate synthase

1. Citrate synthase has been purified from Escherichia coli and shown to exist at an equilibrium between three forms: monomer (mol.wt. 57000), tetramer (mol.wt. 230000) and, possibly, octamer. Modification of the enzyme by photo-oxidation and by treatment with specific chemical reagents has been carried out to gain information on the amino acid residues involved in enzymic activity and in the inhibition of activity by NADH and alpha-oxoglutarate. 2. Several photo-oxidizable amino acids appear to be involved in activity. The nature of the pH-dependence of their rates of photo-oxidation with Methylene Blue suggests that these are histidines, a conclusion supported by the greater rate of photo-inactivation with Rose Bengal and the destruction of activity by diethyl pyrocarbonate. 3. The participation of histidine at the alpha-oxoglutarate effector site is indicated by photo-oxidation and the participation of cysteine at the NADH effector site suggested by photo-oxidation is confirmed by the desensitization to NADH produced by treatment with 5,5'-dithiobis-(2-nitrobenzoate). Inactivation of the enzyme after modification with this reagent suggests the additional involvement of cysteine in catalytic activity. 4. Amino acid analyses of native and photo-oxidized enzyme are consistent with these conclusions. 5. Modification with 2-hydroxy-5-nitrobenzyl bromide indicates the participation of tryptophan in the activity of the enzyme.