An essential histidine residue in the catalytic mechanism of mammalian glutathione reductase

Glutathione reductase from human erythrocytes was inactivated by ethoxyformic anhydride, and > 95% activity was lost by modification of about 1–1.5 histidine residues per flavin (or subunit), as measured by the increased absorbance at 240 nm. Full reactivation was obtained with hydroxylamine. The rate of inactivation increased with pH and an apparent pK = 5.9 was obtained for the protolytic dissociation. The modified enzyme was inactive with NADPH and GSSG as substrates, but almost fully active in catalysis of a transhydrogenase reaction involving pyridine nucleotides. The visible absorption spectrum of oxidized or two-electron-reduced enzyme was not changed, but the flavin fluorescence of oxidized enzyme increased 2-fold after the modification. NADPH or NADP⁺ did not protect the enzyme against inactivation. It is concluded that the modification affects a histidine involved in the second half-reaction of the catalysis, i.e. reduction of GSSG by the dithiol of reduced enzyme. Glutathione reductase from three additional mammalian sources was similarly inactivated, but enzyme from yeast was much less inactivated by the corresponding treatment with ethoxyformic anhydride.     

Triosephosphate isomerase deficiency: a neurodegenerative misfolding disease

A number of neurodegenerative diseases are mediated by mutation-induced protein misfolding. The resulting genetic defects, however, are expressed in varying phenotypes. Of the several well-established glycolytic enzyme deficiencies, triosephosphate isomerase (TPI) deficiency is the only one in which haemolytic anaemia is coupled with progressive, severe neurological disorder. In a Hungarian family with severe decrease in TPI activity, two germ line-identical but phenotypically differing compound heterozygote brothers inherited two independent (Phe(240)-->Leu and Glu(145)-->stop codon) mutations. We have demonstrated recently [Orosz, Oláh, Alvarez, Keserü, Szabó, Wágner, Kovári, Horányi, Baróti, Martial, Hollán and Ovádi (2001) Blood 98, 3106-3112] that the mutations of TPI explain in themselves neither the severe decrease in the enzyme activity characteristic of TPI deficiency nor the enhanced ability of the mutant enzyme from haemolysate of the propositus to associate with subcellular particles. Here we present kinetic (flux analysis), thermodynamic (microcalorimetry and fluores cence spectroscopy), structural (in silico) and ultrastructural (immunoelectron microscopy) data for characterization of mutant isomerase structures and for the TPI-related metabolic processes in normal and deficient cells. The relationships between mutation-induced TPI misfolding and formation of aberrant protein aggregates are discussed.     

A histidine residue in the catalytic mechanism distinguishes Vibrio harveyi aldehyde dehydrogenase from other members of the aldehyde dehydrogenase superfamily

Aldehyde dehydrogenases (ALDHs) catalyze the transfer to NAD(P) of a hydride ion from a thiohemiacetal derivative of the aldehyde coupled with a cysteine residue in the active site. In Vibrio harveyi aldehyde dehydrogenase (Vh-ALDH), a histidine residue (H450) is in proximity (3.8 A) to the cysteine nucleophile (C289) and is thus capable of increasing its reactivity in sharp contrast to other ALDHs in which more distantly located glutamic acid residues are proposed to act as the general base. Mutation of H450 in Vh-ALDH to Gln and Asn resulted in loss of dehydrogenase, (thio)esterase, and acyl-CoA reductase activities; the residual activity of H450Q was higher than that of the H450N mutant in agreement with the capability of Gln but not Asn to partially replace the epsilon-imino group of H450. Coupled with a change in the rate-limiting step, these results indicate that H450 increases the reactivity of C289. Moreover, for the first time, the acylated enzyme intermediate could be directly monitored after reaction with [(3)H]tetradecanoyl-CoA showing that the H450Q mutant was acylated more rapidly than the H450N mutant. Inactivation of the wild-type enzyme with N-ethylmaleimide was much more rapid than the H450Q mutant which in turn was faster than the H450N mutant, demonstrating directly that the nucleophilicity of C289 was affected by H450. As the glutamic acid residue implicated as the general base in promoting cysteine nucleophilicity in other ALDHs is conserved in Vh-ALDH, elucidation of why a histidine residue has evolved to assist in this function in Vh-ALDH will be important to understand the mechanism of ALDHs in general, as well as help delineate the specific roles of the active site glutamic acid residues.     

Is histidine involved in the catalytic mechanism of unspecific carboxylesterases?

The reaction between the liver carboxylesterases from ox and pig and the inhibitor α-bromoacetophenone was studied by amino acid analysis. A significant modification of histidine in pig liver esterase was not found, but there was a slight loss of some other residues. In ox liver esterase the total inhibition correlated with the loss of about 1.7 histidine residues. However, in contrast to previous results with chicken and ox esterases the specific active-site-directed inhibitor E 600 did not prevent the modification of the reactive histidine. It is concluded that an earlier report on the involvement of histidine in the action of liver esterases (1) is partly incorrect or perhaps applicable only to chicken liver esterase.     

The catalytic mechanism of glucose 6-phosphate dehydrogenases: assignment and 1H NMR spectroscopy pH titration of the catalytic histidine residue in the 109 kDa Leuconostoc mesenteroides enzyme

The chemical shifts of the C(epsilon1) and C(delta2) protons of His-240 from the 109 kDa Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) were assigned by comparing 1H and 13C spectra of the wild-type and mutant G6PDs containing the His-240 to asparagine mutation (H240N). Unambiguous assignment of the His-240 1H(epsilon1) resonance was obtained from comparing 13C-1H heteronuclear multiple quantum coherence NMR spectra of wild-type and H240N G6PDs that were selectively labeled with 13C(epsilon1) histidine. The results from NOESY experiments with wild-type and H240N variants were consistent with these assignments and the three-dimensional structure of G6PD. pH titrations show that His-240 has a pK(a) of 6.4. This value is, within experimental error, identical to the value of 6.3 derived from the pH dependence of kcat [Viola, R. E. (1984) Arch. Biochem. Biophys. 228, 415-424], suggesting that the pK(a) of His-240 is unperturbed in the apoenzyme despite being part of a His-Asp catalytic dyad. The results obtained for this 109 kDa enzyme indicate that 1H NMR spectroscopy in combination with heteronuclear methods can be a useful tool for functional analysis of large proteins.     

Crystal structures of human IPP isomerase: new insights into the catalytic mechanism

Type I isopentenyl diphosphate (IPP): dimethylally diphosphate (DMAPP) isomerase is an essential enzyme in human isoprenoid biosynthetic pathway. It catalyzes isomerization of the carbon-carbon double bonds in IPP and DMAPP, which are the basic building blocks for the subsequent biosynthesis. We have determined two crystal structures of human IPP isomerase I (hIPPI) under different crystallization conditions. High similarity between structures of human and Escherichia coli IPP isomerases proves the conserved catalytic mechanism. Unexpectedly, one of the hIPPI structures contains a natural substrate analog ethanol amine pyrophosphate (EAPP). Based on this structure, a water molecule is proposed to be the direct proton donor for IPP and different conformations of IPP and DMAPP bound in the enzyme are also proposed. In addition, structures of human IPPI show a flexible N-terminal alpha-helix covering the active pocket and blocking the entrance, which is absent in E. coli IPPI. Besides, the active site conformation is not the same in the two hIPPI structures. Such difference leads to a hypothesis that substrate binding induces conformational change in the active site. The inhibition mechanism of high Mn(2+) concentrations is also discussed.     

The electrophilic and leaving group phosphates in the catalytic mechanism of yeast pyrophosphatase

Binding of pyrophosphate or two phosphate molecules to the pyrophosphatase (PPase) active site occurs at two subsites, P1 and P2. Mutations at P2 subsite residues (Y93F and K56R) caused a much greater decrease in phosphate binding affinity of yeast PPase in the presence of Mn(2+) or Co(2+) than mutations at P1 subsite residues (R78K and K193R). Phosphate binding was estimated in these experiments from the inhibition of ATP hydrolysis at a sub-K(m) concentration of ATP. Tight phosphate binding required four Mn(2+) ions/active site. These data identify P2 as the high affinity subsite and P1 as the low affinity subsite, the difference in the affinities being at least 250-fold. The time course of five "isotopomers" of phosphate that have from zero to four (18)O during [(18)O]P(i)-[(16)O]H(2)O oxygen exchange indicated that the phosphate containing added water is released after the leaving group phosphate during pyrophosphate hydrolysis. These findings provide support for the structure-based mechanism in which pyrophosphate hydrolysis involves water attack on the phosphorus atom located at the P2 subsite of PPase.     

Identification of the catalytic histidine residue participating in the charge-relay system of carboxypeptidase Y.

The essential histidine residue of carboxypeptidase Y (CPY) was modified by a site-specific reagent, a chloromethylketone derivative of benzyloxycarbonyl-L-phenylalanine. The single modified histidine residue was converted to N tau-carboxy-methyl histidine (cmHis) upon performic acid oxidation. A peptide containing cmHis was isolated from the tryptic-thermolytic digest. Based on the amino acid composition and sequence analysis, the peptide is shown to be Val-Phe-Asp-Gly-Gly-cmHis-MetO2-Val-Pro, which was derived from CPY cleaved by trypsin at Arg 391 and thermolysin at Phe 401, and thus His 397 was modified. This histidine residue has been implicated previously by X-ray analysis to participate in the charge-relay system of CPY.     

Conformational change in the vinculin C-terminal depends on a critical histidine residue (His-906)

A phospholipid-controlled interaction between the N-terminal and C-terminal domains of vinculin is thought to be a major mechanism that regulates binding activities of the protein. To probe the mechanisms underlying these interactions we used chemical modification and site-directed mutagenesis directed at histidine residues. Diethylpyrocarbonate (DEPC) modification of the C-terminal, but not the N-terminal, domain greatly decreased affinity of the N-terminal-C-terminal binding, implicating histidine residues in the C-terminal. Mutation of either or both C-terminal histidines (at positions 906 and 1026), however, did not affect N-C binding at neutral pH. The H906A mutation did prevent DEPC effects and also prevented the normal decrease in binding affinity for the N-terminal at lower pH. We found that the wild type C-terminal domain, but not the H906A mutant, underwent a conformational change at pH 6.5, reflected in an altered circular dichroism spectrum and apparent oligomerization. Phospholipid also induced conformational changes in the wild type C-terminal domain but not in the H906A mutant, even though the mutant protein did bind to the phospholipid. Finally, the sensitivity of the N-C interaction to phospholipid was much reduced by the H906A mutation. These results show that H906 plays a key role in the conformational dynamics of the C-terminal domain and thus the regulation of vinculin.     

Importance of histidine residue 25 of rat heme oxygenase for its catalytic activity.

A truncated, soluble, and enzymatically active rat heme oxygenase lacking its membrane-associative, C-terminal segment was expressed in E. coli strain JM109. The roles of its four histidine residues were examined by determining the enzymatic activities of mutant enzymes in which each of these residues in turn was replaced by alanine. Mutation of histidine residue 25 to alanine resulted in marked decrease in activity for heme breakdown, indicating that this histidine residue has an important role in the heme oxygenase reaction.     

pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pK_a values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pK_a values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.     

Structural basis for phosphomannose isomerase activity in phosphoglucose isomerase from Pyrobaculum aerophilum: a subtle difference between distantly related enzymes

The crystal structure of a dual-specificity phosphoglucose/phosphomannose isomerase from the crenarchaeon Pyrobaculum aerophilum (PaPGI/PMI) has been determined in complex with glucose 6-phosphate at 1.16 A resolution and with fructose 6-phosphate at 1.5 A resolution. Subsequent modeling of mannose 6-phosphate (M6P) into the active site of the enzyme shows that the PMI activity of this enzyme may be due to the additional space imparted by a threonine. In PGIs from bacterial and eukaryotic sources, which cannot use M6P as a substrate, the equivalent residue is a glutamine. The increased space may permit rotation of the C2-C3 bond in M6P to facilitate abstraction of a proton from C2 by Glu203 and, after a further C2-C3 rotation of the resulting cis-enediolate, re-donation of a proton to C1 by the same residue. A proline residue (in place of a glycine in PGI) may also promote PMI activity by positioning the C1-O1 region of M6P. Thus, the PMI reaction in PaPGI/PMI probably uses a cis-enediol mechanism of catalysis, and this activity appears to arise from a subtle difference in the architecture of the enzyme, compared to bacterial and eukaryotic PGIs.     

On the three-dimensional structure and catalytic mechanism of triose phosphate isomerase

Triose phosphate isomerase is a dimeric enzyme of molecular mass 56 000 which catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate. The crystal structure of the enzyme from chicken muscle has been determined at a resolution of 2.5 A, and an independent determination of the structure of the yeast enzyme has just been completed at 3 A resolution. The conformation of the polypeptide chain is essentially identical in the two structures, and consists of an inner cylinder of eight strands of parallel beta-pleated sheet, with mostly helical segments connecting each strand. The active site is a pocket containing glutamic acid 165, which is believed to act as a base in the reaction. Crystallographic studies of the binding of DHAP to both the chicken and the yeast enzymes reveal a common mode of binding and suggest a mechanisms for catalysis involving polarization of the substrate carbonyl group.     

Triosephosphate isomerase by consensus design: dramatic differences in physical properties and activity of related variants

Consensus design, the selection of mutations based on the most common amino acid in each position of a multiple sequence alignment, has proven to be an efficient way to engineer stabilized mutants and even to design entire proteins. However, its application has been limited to small motifs or small families of highly related proteins. Also, we have little idea of how information that specifies a protein's properties is distributed between positional effects (consensus) and interactions between positions (correlated occurrences of amino acids). Here, we designed several consensus variants of triosephosphate isomerase (TIM), a large, diverse family of complex enzymes. The first variant was only weakly active, had molten globular characteristics, and was monomeric at 25 °C despite being based on nearly all dimeric enzymes. A closely related variant from curation of the sequence database resulted in a native-like dimeric TIM with near-diffusion-controlled kinetics. Both enzymes vary substantially (30–40%) from any natural TIM, but they differ from each other in only a relatively small number of unconserved positions. We demonstrate that consensus design is sufficient to engineer a sophisticated protein that requires precise substrate positioning and coordinated loop motion. The difference in oligomeric states and native-like properties for the two consensus variants is not a result of defects in the dimerization interface but rather disparate global properties of the proteins. These results have important implications for the role of correlated amino acids, the ability of TIM to function as a monomer, and the ability of molten globular proteins to carry out complex reactions.     

Evidence for the essential histidine residue at the active site of glucose/xylose isomerase from Streptomyces

Modification of glucose/xylose isomerase from Streptomyces sp. NCIM 2730 by diethylpyrocarbonate (DEPC) or its photo-oxidation in presence of rose bengal or methylene blue caused rapid loss in its activity. The inactivation of the enzyme was accompanied by an increase in the absorbance at 240 nm and was reversed by hydroxylamine. Glucose and xylose but not Mg++ and Co++ afforded significant protection to the enzyme from inactivation by DEPC. Inactivation followed pseudo-first-order kinetics and modification of a single histidine residue per mole of enzyme was indicated.     

Solution structure and catalytic mechanism of human protein histidine phosphatase 1

Protein histidine phosphorylation exists widely in vertebrates, and it plays important roles in signal transduction and other cellular functions. However, knowledge about eukaryotic PHPT (protein histidine phosphatase) is still very limited. To date, only one vertebrate PHPT has been discovered, and two crystal structures of hPHPT1 (human PHPT1) have been solved. However, these two structures gave different ligand-binding sites and co-ordination patterns. In the present paper, we have solved the solution structures of hPHPT1 in both P(i)-free and P(i)-bound states. Through comparison of the structures, along with a mutagenesis study, we have determined the active site of hPHPT1. In contrast with previous results, our results indicate that the active site is located between helix alpha1 and loop L5. His(53) was identified to be the catalytic residue, and the NH groups of residues His(53), Ala(54) and Ala(96) and the OH group of Ser(94) should act as anchors of P(i) or substrate by forming H-bonds with P(i). On the basis of our results, a catalytic mechanism is proposed for hPHPT1: the imidazole ring of His(53) serves as a general base to activate a water molecule, and the activated water would attack the substrate as a nucleophile in the catalysis; the positively charged side chain of Lys(21) can help stabilize the transition state. No similar catalytic mechanism can be found in the EzCatDB database.     

Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from [1(R)-3H]dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the "exchange-conversion" experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase.(ABSTRACT TRUNCATED AT 250 WORDS)