Active Center Histidine Research Articles

The Crystal Structure of Human Transketolase and New Insights into Its Mode of Action

The crystal structure of human transketolase (TKT), a thiamine diphosphate (ThDP) and Ca(2+)-dependent enzyme that catalyzes the interketol transfer between ketoses and aldoses as part of the pentose phosphate pathway, has been determined to 1.75 Å resolution. The recombinantly produced protein crystallized in space group C2 containing one monomer in the asymmetric unit. Two monomers form the homodimeric biological assembly with two identical active sites at the dimer interface. Although the protomer exhibits the typical three (α/β)-domain structure and topology reported for TKTs from other species, structural differences are observed for several loop regions and the linker that connects the PP and Pyr domain. The cofactor and substrate binding sites of human TKT bear high resemblance to those of other TKTs but also feature unique properties, including two lysines and a serine that interact with the β-phosphate of ThDP. Furthermore, Gln(189) spans over the thiazolium moiety of ThDP and replaces an isoleucine found in most non-mammalian TKTs. The side chain of Gln(428) forms a hydrogen bond with the 4'-amino group of ThDP and replaces a histidine that is invariant in all non-mammalian TKTs. All other amino acids involved in substrate binding and catalysis are strictly conserved. Besides a steady-state kinetic analysis, microscopic equilibria of the donor half-reaction were characterized by an NMR-based intermediate analysis. These studies reveal that formation of the central 1,2-dihydroxyethyl-ThDP carbanion-enamine intermediate is thermodynamically favored with increasing carbon chain length of the donor ketose substrate. Based on the structure of human transketolase and sequence alignments, putative functional properties of the related transketolase-like proteins TKTL1 and -2 are discussed in light of recent findings suggesting that TKTL1 plays a role in cancerogenesis.

Direct proton magnetic resonance determination of the pKa of the active center histidine in thiolsubtilisin.

The serine proteases constitute a group of endopeptidases whose members owe their catalytic activity to the presence of a catalytic triad of amino acids consisting of a serine, a histidine and an aspartate. The pK(a) values for this histidine have been determined for several cases in which there is a negative charge installed at the serine to mimic the oxyanionic intermediate and related transition state for the catalytic pathway. Instances from this laboratory include (1) replacement of the serine by a cysteine in subtilisin to create a thiolate; (2) formation of monoisopropylphosphoryl-Ser 195 monoanionic phosphodiesters (in trypsin and chymotrypsin, Ser 221 in subtilisins); and (3) tetrahedral boronates formed with peptide boronic acids. The nuclear magnetic resonance (NMR) signals pertinent to this histidine, or signals indirectly reflecting the state of ionization of this histidine, have been used effectively to monitor changes in the active center ionization state. In every case studied, there is elevation of the pK(a) at the histidine when the negative charge is installed at the serine position. Herein is reported the first NMR measurement of the active center His 63 pK(a) in thiolsubtilisin Carlsberg; it is elevated by 3 units compared with the parent enzyme. Using a numerical solution (finite difference) of the Poisson-Boltzmann equation, a protein dielectric constant of 4 provides a good estimate of the experimentally observed pK(a) elevations. Very significantly, a very low protein dielectric constant (epsilon(p) = 3-5) is required in all of the comparisons, and for all three enzymes used (chymotrypsin, trypsin, and subtilisin). Finally, we discuss why the electrostatic perturbation sensed at His of the active center is more amplified by a negative charge on the Ser side than the same charge on the Asp side. A plausible explanation is that the positive charge on the imidazolium ring of the His is localized, with the N(delta 1) carrying a smaller fraction, the N(epsilon 2) carrying the bulk of the positive charge.

Assignment of the Nε2H and Nδ1H Resonances at the Active-Center Histidine in Chymotrypsin and Subtilisin Complexed to Peptideboronic Acids without Specific 15N Labeling1

A combination of 1H and 15N nuclear magnetic resonance experiments have been carried out to assign the two high-frequency 1H resonances that result from the complexation of subtilisin E and MeoSuc-Ala-Ala-Pro-boroPhe, a potent peptideboronic acid inhibitor of both subtilisins from a variety of sources and chymotrypsin. First, it was demonstrated unequivocally using two auxotrophs of Bacillus subtilis that the proton resonances at 16 and 17 ppm pertain to a histidine residue. Next it was shown by both 1D and 2D methods that the two proton resonances pertain to the same histidine. Finally, in the subtilisin E-peptideboronate complex, all of the imidazole proton and nitrogen resonances pertinent to this His64 were assigned as follows: Ne2 at 183 and Nδ1 at 189 ppm; Ne2H at 16 and Nδ1H at 17.4 ppm; Ce1H at 9.20 and Cδ2H at 7.09 ppm. Using the 1D NOE method demonstrated on the subtilisin−peptideboronate complex, the resonances due to complexation were also assigned in the chymotrypsin−peptideboronate complex....

Low Barrier Hydrogen Bond Is Absent in the Catalytic Triads in the Ground State but Is Present in a Transition-state Complex in the Prolyl Oligopeptidase Family of Serine Proteases

High frequency proton NMR spectra for two members of the prolyl oligopeptidase class of serine proteases, prolyl oligopeptidase and oligopeptidase B, showed that resonances corresponding to the active center histidine Ndelta1H and Nepsilon2H generally observed in this region, are absent in these enzymes. However, for both enzymes, as well as with the H652A and H652Q active center variants of oligopeptidase B, there are two resonances observed in this region that could be assigned to two protonated histidines with a noncatalytic function. The results indicate that these two histidines participate in strong hydrogen bonds. The absence of resonances pertinent to the active center histidine resonances suggests the absence of a low barrier hydrogen bond between the Asp and His in these two enzymes in their ground states. Addition of the peptide boronic acid t-butoxycarbonyl-(D)Val-Leu-(L)boroArg to oligopeptidase B resulted in potent, slow binding inhibition of the enzyme and the appearance of a new resonance at 15.8 ppm, whose chemical shift is appropriate for a tetrahedral boronate complex and a low barrier hydrogen bond. The results demonstrate important dissimilarities between the active centers of the prolyl oligopeptidase class of serine proteases and the pancreatic and subtilisin classes both in the ground state and in the transition-state analog complexes.

Cloning and Functional Analysis of the ndk1 Gene Encoding Nucleoside-diphosphate Kinase in Schizosaccharomyces pombe

We cloned the ndk1 gene encoding a subunit of nucleoside-diphosphate kinase (NDK) from Schizosaccharomyces pombe, by using polymerase chain reaction. The deduced ndk1 gene product has 151 amino acid residues and is approximately 60% identical with both Saccharomyces cerevisiae and mammalian NDKs. The gene product exhibited NDK activity and cross-reacted with antibodies raised against rat NDK. Disruption of ndk1 greatly reduced the cellular NDK activity but caused no obvious phenotype in cell growth and sexual development of the organism. However, a mutated allele of ndk1 could inhibit sexual development in a dominant-negative manner. This allele carried a point mutation in cysteine 116, which locates next to the putative active center histidine 117, and the mutant gene product showed no NDK activity. Gene expression inducible in response to mating pheromone signaling was decreased in cells carrying the dominant-negative allele. Cases have been reported in higher eukaryotes in which NDK appears to play a more sophisticated role than a simple catalyst in cell physiology, and the results of this study suggest that S. pombe NDK may also perform such a role in regulation of sexual development in the fission yeast.

Proton magnetic resonance studies of the active center histidine of chymotrypsin complexed to peptideboronic acids: Solvent accessibility to the N.delta. and N.epsilon. sites can differentiate slow-binding and rapidly reversible inhibitors

Proton magnetic resonance studies of the active center histidine of chymotrypsin complexed to peptideboronic acids: Solvent accessibility to the N.delta. and N.epsilon. sites can differentiate slow-binding and rapidly reversible inhibitors

Methylation of the active center histidine 217 in D-amino acid oxidase by methyl-p-nitrobenzenesulfonate.

Incubation of D-amino acid oxidase with excess methyl-p-nitrobenzenesulfonate results in a pseudo-first order, irreversible loss of 95% of the assayable activity using D-phenylglycine as substrate. The rate of inactivation reaches a limiting value of 0.021 min-1 (pH 7.7, 22 degrees C) as the concentration of inhibitor is increased, consistent with a two-step mechanism with complex formation prior to covalent inactivation. This rate decreases with decreasing pH in a manner that suggests that the basic form of a group within the enzyme, with an apparent pKa of 6.7, is required for inactivation. Approximately three [14C]methyl residues are covalently incorporated at maximal inactivation. The competitive inhibitor benzoate prevents inactivation and the incorporation of approximately 1 methyl residue. Histidine 217, the residue modified by 5-dimethylaminonaphthalene-1-sulfonyl chloride (Swenson, R. P., Williams, C. H., Jr., and Massey, V. (1983) J. Biol. Chem. 258, 497-502), was identified as the major residue modified in the absence of benzoate. Both the 1-methyl and 3-methyl derivatives of histidine 217 were observed in a mole ratio of 0.64:0.36.

Phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus: factor IIIlac, a trimeric phospho-carrier protein that also acts as a phase transfer catalyst.

Factor IIIlac (FIII) consists of three identical subunits. It could be shown that each of the subunits carries a phosphoryl group upon phosphorylation (P-FIII) with phosphoenolpyruvate (PEP), enzyme I, and histidine-containing phospho-carrier protein (HPr). The phosphoryl group is bound to a histidyl residue in P-FIII. Each subunit of FIII contains four histidyl residues. After tryptic cleavage a peptide was isolated that contained one other histidyl residue besides the active center histidine. By further cleavage of the peptide T-2 with V-8 Staphylococcus aureus protease it could be shown that His-19 in the sequence of the peptide T-2 is the active center histidine. Another peptide (1-38), caused by incomplete tryptic cleavage, could be isolated. It inhibited the phospho-transfer reaction from PEP to the sugar molecule at the step of factor III-enzyme II recognition. It competes with factor III for the binding site of enzyme II, the membrane component. It is a very hydrophobic peptide. This hydrophobic region is buried in factor III. But upon phosphorylation of factor III it is turned out. Thus P-FIII binds to Triton X-100 micelles whereas factor III does not. This conformational change caused by phosphorylation could be shown by proton nuclear magnetic resonance methods [Kalbitzer, H.R., Deutscher, J., Hengstenberg, W., & Rösch, P. (1981) Biochemistry 20, 6178-6185], by circular dichroism spectroscopy, and by the Ouchterlony double-diffusion method. Antibodies against FIII do not precipitate P-FIII.

The structural isomerisation of human-muscle adenylate kinase as studied by 1H-nuclear magnetic resonance.

Human muscle adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3.) was studied by 1H-nuclear magnetic resonance spectroscopy. The C-2 and C-4 proton resonances of the active-center histidine His-36 could be identified; the pK of His-36 was determined as 6.1. The pK of His-189 is very low (4.9) although it is located at the surface of the protein. Other resonance lines are discussed in comparison with NMR spectra of porcine adenylate kinase [McDonald et al. (1975) J. Biol. Chem. 250, 6947-6954]. A pH-dependent structural isomerization as shown by X-ray crystallography in the pig enzyme [Pai et al. (1977) J. Mol. Biol. 114, 37-45] was not observed for human adenylate kinase in solution. However, the binding of adenosine(5')pentaphospho(5')adenosine (Ap5A), a bisubstrate inhibitor, to adenylate kinase causes an overall change of the NMR spectrum indicative of a large conformational change of the enzyme. The exchange rate (koff) for Ap5A was estimated as 10 s-1 and decreases by addition of Mg2+. On the basis of these values and the known dissociation constant it is likely that the binding of Ap5A is a diffusion-controlled process kon being 10(8) M-1 s-1. In conclusion, the system Ap5A/Mg2+/human adenylate kinase, which has been studied by NMR spectroscopy and X-ray diffraction in parallel, is suitable for analyzing the induced fit postulated by Jencks for all kinase-catalyzed reactions.

Active subunits in hybrid-modified malate dehydrogenase.

Inactivation of porcine heart mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) by selective modification of an active center histidine residue with the reagent iodoacetamide has been further investigated to examine the existence of and the enzymatic activity of a hybrid (half)-modified dimer. The loss of enzymatic activity during iodo(1-14C) acetamide modification is linear with 14C incorporation. Enzyme was modified to various extents and the reaction was quenched. Microzonal electrophoresis was performed to separate native dimeric enzyme, hybrid-modified enzyme, and doubly modified enzyme. The distribution of each species was quantitated by scanning densitometry. The distribution generated throughout the time course of inactivation indicates that both subunits are modified independently and at the same rate. It is apparent that the hybrid-modified dimer contributes one-half of the enzymatic activity of a native dimer in the standard assay. Kinetic studies were performed and the results indicate that there is no apparent change in kinetic parameters between a subunit of the native dimer and the active subunit in the hybrid-modified dimer. Dissociation and reassociation of a mixture of native enzyme and doubly-iodoacetamide-modified enzyme indicates that there is no preferential association of a modified subunit with another modified subunit, or of a native subunit with another native subunit, but rather, association is random with respect to native and iodoacetamide-modified subunits.

Phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus: 1H nuclear magnetic resonance studies on phosphorylated and unphosphorylated factor IIIlac and its interaction with the phosphocarrier protein HPr.

The trimeric phosphocarrier protein factor III specific for galactosides was investigated by 1H NMR spectroscopy. The protomer contains four histidyl residues with acidic pK values in the range 5.6-6.2. One of the histidyl residues, His-B, carries the phosphoryl group. The pK value of His-B increases from 6.0 to 8.6 upon phosphorylation. To determine the position of the phosphoryl group with respect to the nitrogens required the isolation of a peptide T-2 containing the phosphorylated active-center histidine and one of the other histidines. The pK value and the chemical shift of the phosphopeptide clearly indicated the phosphorus to be bound to the N-3 atom of the imidazole ring. The temperature dependence of the factor III spectrum demonstrates multiple conformations which exchange rapidly on the NMR time scale. Titration of factor III with HPr protein showed an upfield shift of the active-center histidine, indicating complex formation between both proteins. Phosphorylation of both proteins abolished the interaction, which is plausible from mechanistic considerations.

Studies on the Biochemistry of Urokinase

SummaryDirect evidence for an active center histidine residue in urokinase (UK) was obtained with use of newly synthesized peptide chloroketones Ac-Gly-Lys-CH2C1 and Nle-Gly-Lys-CH2C1. Stoichiometric inactivation by DFP provided further evidence that UK is a serine protease. Essential histidine and serine residues were both located in the heavy chain of the 47,0 M. W.UK. The high M.W. form can be converted (catalytically) to the low M. W. form.9 partially purified human urinary UK preparations (5 with predominantly high M. W. UK), varying in purity and proportion of high and low M. W. forms, were found to be heterogeneous by a number of acrylamide electrophoretic procedures. 7 preparations had strikingly similar molar activities at excess substrate, except for the lower values found in 2 predominantly high M. W. UK preparations from the same supplier. 2 high M. W. UK preparations from another supplier showed a definite increase in activity when assayed at low plasminogen concentration, but this effect was abolished after gel filtration (Sephadex G-25), by further purification with affinity chromatography, or when assayed with excess plasminogen.The high and low M. W. forms of UK (47,000 and 33,400 M. W.), isolated and purified by Sepharose-EACA-agmatine affinity chromatography were shown to be homogeneous by Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis (PAGE) and by 14C-DFP and 14C-NPGB incorporation before SDS-PAGE. Comparative properties of the high M. W. vs low M. W. forms were as follows: specific activity (104,000 IU/mg vs 226,000) ; 2 chains (33,100 and 18,600 M.W.) linked by disulfide(s) vs a single chain; pi 8.60 (major subform) and pi 8.90 (minor subform) vs pi 8.35, 8.60, 8.70 (major subform) and pi 8.05 (minor subform); and second order kinetics for DFP inactivation (400 vs 770 M−1 min−1). The molar activities were similar (9.6 × 109 and 10.2 × 109IUm/mole) for each form.

The amino acid sequence of a peptide containing the active center histidine residue of porcine malate dehydrogenase — a correction

The amino acid sequence of a peptide containing the active center histidine residue of porcine malate dehydrogenase — a correction

Characterization of porcine malate dehydrogenase: II. Amino acid sequence of a peptide containing the active center histidine residue

Selective chemical modification of 2 residues of histidine in mitochondrial malate dehydrogenase ( l-malate:NAD + oxidoreductase, EC 1.1.1.37) from porcine heart with the reagent iodoacetamide occurs concomitant with total inactivation of the enzyme. The presence of the reduced cofactor NADH fully protects the enzyme from inactivation, thus suggesting that alkylation is occuring at or near the coenzyme binding site. A tryptic peptide containing the modified residue has been isolated and analyzed for composition. The amino acid sequence of this peptide has been determined to be NH 2-Val-Ser-Val-Pro-Ile-carboxymethylHis-Gly-Gly-(Val,Ala,Gly)-Lys-OH. The hydrophobic nature of the peptide, in relation to its function in the area of the coenzyme binding site, is discussed.

The Active Center of Aspartate Transaminase: A FLUORINE-19 NUCLEAR MAGNETIC RESONANCE STUDY OF THE ANION BINDING SITE

Abstract The binding of trifluoroacetate anion to aspartate transaminase (EC 2.6.1.1) has been measured by the changes of the chemical shift in the 19F nuclear magnetic resonance spectrum. The dissociation constants for the enzyme-trifluoroacetate complex are pH dependent with values ranging from 15 to 36 mm at pH values between 5 and 8. Stoichiometry shows that there is only one anion binding site. The pH-dependence of trifluoroacetate binding indicates that a group in the enzyme with a pKa of 6.2 ± 0.2 is responsible for anion binding. Apoenzyme and holoenzyme after reduction of the pyridoxyl-lysine aldimine with NaBH4 (reduced enzyme), show the same pH dependence pattern and the same pKa value for trifluoroacetate binding as native enzyme. Controlled photooxidation of the active site histidine residue results in the elimination of the anion binding site. Chloride and succinate compete with trifluoroacetate for the same site in the native, apoenzyme, and reduced enzymes. On the other hand, the pseudosubstrate α-methylaspartate, which binds covalently to the enzyme to form the initial aldimine enzyme-substrate complex, competes with trifluoroacetate for the binding site only in the native enzyme, but this does not occur after the removal or reduction of the active site Schiff's base, i.e. in the apoenzyme and reduced enzyme. The positively charged anion binding site in the protein is postulated to be the active site histidine. A model is proposed for mutual exclusion between anion and substrate from the active center without a sharing of common subsites. The model is consistent with the stoichiometry, competition effects and pH dependence of ligand binding, as well as with the elimination of anion binding after photooxidation of the active center histidine without a subsequent effect on substrate affinity.

The Purification of Thrombin and Isolation of a Peptide Containing the Active Center Histidine

A chromatographic purification of bovine thrombin from commercial starting material is described which yields preparations judged to be essentially pure, that is, with a clotting activity of 2100 to 2500 NIH units per mg. In addition to the two-chain form of thrombin, the purified enzyme contains a three-chain form arising from cleavage of the B chain at arginine-73 (or 76). This form must be fully active since the specific clotting activity of various preparations does not vary with the content of the three-chain form, amounting at times to 70%. Inactivation of thrombin with Nα-tosyl lysyl chloromethyl ketone and Nα-(p-nitrobenzyloxycarbonyl)arginyl chloromethyl ketone results in alkylation of nitrogen-3 of the active center histidine with loss of both clotting and esterase activities. A radioactive peptide has been isolated from thrombin inactivated with tritiated Nα-tosyl lysyl chloromethyl ketone and shown to contain histidine-43.

Amino acids around an active center histidine in a turtle chymotrypsin

Amino acids around an active center histidine in a turtle chymotrypsin

Evidence for an Active-Center Histidine in Trypsin through Use of a Specific Reagent, 1-Chloro-3-tosylamido-7-amino-2-heptanone, the Chloromethyl Ketone Derived from Nα-Tosyl-L-lysine*

Evidence for an Active-Center Histidine in Trypsin through Use of a Specific Reagent, 1-Chloro-3-tosylamido-7-amino-2-heptanone, the Chloromethyl Ketone Derived from N^α-Tosyl-L-lysine^*

An Active Center Histidine Peptide of α-Chymotrypsin

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAn Active Center Histidine Peptide of α-ChymotrypsinEng Bee. Ong, Elliott. Shaw, and Guenther. SchoellmannCite this: J. Am. Chem. Soc. 1964, 86, 6, 1271–1272Publication Date (Print):March 1, 1964Publication History Published online1 May 2002Published inissue 1 March 1964https://doi.org/10.1021/ja01060a081RIGHTS & PERMISSIONSArticle Views62Altmetric-Citations55LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (296 KB) Get e-Alerts Get e-Alerts