A kinetic study on the chemical cleavage of nucleoside diphosphate sugars

Nucleoside diphosphate sugars serve in essential roles in metabolic processes. They have, therefore, been used in mechanistic studies on glycosylation reactions, and their analogues have been synthesised as enzyme and receptor inhibitors. Despite extensive biochemical research, little is known about their chemical reactions. In the present work the chemical cleavage of two different types of nucleoside diphosphate sugars has been studied. UDP-Glc is phosphorylated at the anomeric carbon, whereas in ADP-Rib C-1 is unsubstituted, allowing hence the equilibrium between cyclic hemiacetal and acyclic carbonyl forms. Due to the structural difference, these substrates react via different pathways under slightly alkaline conditions: while UDP-Glc reacts exclusively by a nucleophilic attack of a glucose hydroxyl group on the diphosphate moiety, ADP-Rib undergoes a complex reaction sequence that involves isomerisation processes of the acyclic ribose sugar and results in a release of ADP.     

Identification of a critical lysine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cell. Comparison with the rabbit muscle enzyme.

The involvement of the lysine residue present at the active site of Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (Gra3PDH) was investigated by using the lysine specific reagents trinitrobenzenesulfonic acid (TNBS) and pyridoxal phosphate (PP). Both TNBS and PP inactivated EAC cell Gra3PDH with pseudo-first-order kinetics with the rate dependent on modifier concentration. Kinetic analysis, including a Tsou plot, indicated that both TNBS and PP apparently react with one lysine residue per enzyme molecule. Two of the substrates, d-glyceraldehyde-3-phosphate and NAD, and also NADH, the product and competitive inhibitor, almost completely protected the enzyme from inactivation by TNBS. A comparative study of Gra3PDH of EAC cell and rabbit muscle indicates that the nature of active site of the enzyme is significantly different in these two cells. A double inhibition study using 5,5'-dithiobis(2-nitrobenzoic acid) and TNBS and subsequent reactivation of only the rabbit muscle enzyme by dithiothreitol suggested that a cysteine residue of this enzyme possibly reacts with TNBS. These studies on the other hand, confirm that an essential lysine residue is involved in the catalytic activity of the EAC cell enzyme. This difference in the nature of the active site of EAC cell Gra3PDH that may be related to the high glycolysis of malignant cells has been discussed.     

Uridine diphosphate release mechanism in O-N-acetylglucosamine (O-GlcNAc) transferase catalysis

O-linked N-acetylglucosamine transferase (OGT) is an essential enzyme that catalyzes the covalent bonding of N-acetylglucosamine (GlcNAc) to the hydroxyl group of a serine or threonine in the target protein. It plays an important role in many important cellular physiological catalytic reactions. Here, we determine the binding mode and the binding free energy of the OGT product (uridine diphosphate, UDP) as well as the hydrogen-bond-dependent release mechanism using extensive molecular dynamic simulations. The Lys634, Asn838, Gln839, Lys842, His901, and Asp925 residues were identified to play a major role in the UDP stabilization in the active site of OGT, where hydrogen bonding and π-π interactions mainly occur. The calculations on the mutant forms support our results. Sixteen possible release channels were identified while the two most favorable channels were determined using random acceleration molecular dynamics (RAMD) simulations combined with the constant velocity pulling (PCV) method. The thermodynamic and dynamic properties as along with the corresponding mechanism were determined and discussed according to the umbrella sampling technique. For the most optimal channel, the main free energy barrier is 13?kcal/mol, which probably originates from the hydrogen bonds between UDP and the Ala896 and Asp925 residues. Moreover, the unstable hydrogen bonds and the rollback of the ligand likely cause the other two small obstacles. This work clarifies the ligand transport mechanism in the OGT enzymatic process and is a great resource for designing inhibitors based on UDP or UDP-GlcNAc.     

Structures of trihydroxynaphthalene reductase-fungicide complexes: implications for structure-based design and catalysis

BACKGROUND: Trihydroxynaphthalene reductase catalyzes two intermediate steps in the fungal melanin biosynthetic pathway. The enzyme, a typical short-chain dehydrogenase, is the biochemical target of three commercial fungicides. The fungicides bind preferentially to the NADPH form of the enzyme. RESULTS: Three X-ray structures of the Magnaporthe grisea enzyme complexed with NADPH and two commercial and one experimental fungicide were determined at 1.7 A (pyroquilon), 2.0 A (2,3-dihydro-4-nitro-1H-inden-1-one, 1), and 2.1 A (phthalide) resolutions. The chemically distinct inhibitors occupy similar space within the enzyme's active site. The three inhibitors share hydrogen bonds with the side chain hydroxyls of Ser-164 and Tyr-178 via a carbonyl oxygen (pyroquilon and 1) or via a carbonyl oxygen and a ring oxygen (phthalide). Active site residues occupy similar positions among the three structures. A buried water molecule that is hydrogen bonded to the NZ nitrogen of Lys-182 in each of the three structures likely serves to stabilize the cationic form of the residue for participation in catalysis. CONCLUSIONS: The pro S hydrogen of NADPH (which is transferred as a hydride to the enzyme's naphthol substrates) is directed toward the carbonyl carbon of the inhibitors that mimic an intermediate along the reaction coordinate. Modeling tetrahydroxynaphthalene and trihydroxynaphthalene in the active site shows steric and electrostatic repulsion between the extra hydroxyl oxygen of the former substrate and the sulfur atom of Met-283 (the C-terminal residue), which accounts, in part, for the 4-fold greater substrate specificity for trihydroxynaphthalene over tetrahydroxynaphthalene.     

LDH and structural analogues of pyruvate (author's transl)]

Pyruvate related compounds have been tested for their active or inhibitory properties on LDH. Special structure features were needed for compounds to be bound to the enzyme active site. Pyruvate and the LDH molecule were bound by the interaction of the carboxyl group or its ester derivative, with the enzyme. Ethyl pyruvate seemed to behave as a substrate of the enzyme whereas acetophenone, ethyl acetoacetate and beta-oxoglutarate did not act as substrates or inhibitors of LDH. Phenyl pyruvate, alpha-oxoglutarate and L-mandelate are not substrates but inhibitors of the LDH. It seems that a structure having a carbonyl group in the alpha-position to the carboxyl is required for the binding of a compound to the LDH molecule. Glyoxylate alpha-oxobutyrate and alpha-oxovalerianate appear to be worse LDH substrates than pyruvate itself. This seems to suggest that the shortening or the extension of the pyruvate aliphatic chain induces a decrease of the affinity of the enzyme towards their substrate homologues.     

Characterization of the mouse sperm plasma membrane zona-binding site sensitive to trypsin inhibitors

The first contact of mammalian gametes is the binding of the spermatozoon to the zona pellucida of the egg. Previous work has shown that binding of the spermatozoon to the zona in the mouse occurs prior to the acrosome reaction and that trypsin inhibitors block this initial binding. This suggests that the sperm surface contains a trypsinlike binding site that functions by an active site mechanism to effect initial zona binding. When suspensions of twice-washed spermatozoa were incubated with the serine protease active site titrant, 4-methylumbelliferyl p-guanidinobenzoate (MUGB), the titrant was hydrolyzed at a rate of 8 pmoles/min-10(6) cells. MUGB was found to inhibit the binding of spermatozoa to the zona pellucida. The degree of inhibition and the rate of hydrolysis of MUGB by washed spermatozoa depend on the concentration of titrant, with half maximal effects at 13 microM and a linear correlation with r = 0.99. The analogous lysyl and arginyl trypsin substrates containing 7-amino-4-methylcoumarin as the fluorogenic leaving group were not hydrolyzed under the same conditions and did not inhibit zona binding. Both binding of sperm to zona-intact eggs and the hydrolysis of MUGB by sperm are inhibited by p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, and acid-solubilized zonae. The linear correlation coefficients of the inhibition of sperm binding and MUGB hydrolysis by these three substances are greater than 0.92. This "trypsinlike" sperm site is essential for sperm binding to the zona: its stereospecificity is unique in that it reacts with trypsin inhibitors but not with trypsin substrates.     

Chemical modification of lysine residues in bacterial formate dehydrogenase

Specific modification of 4.4 lysine residues per molecule of formate dehydrogenase, from the methylotrophic bacterium Achromobacter parvulus I by pyridoxal, results in complete inactivation of the enzyme. The concentration effect of the modifying agent and substrates on the inactivation of formate dehydrogenase has been studied. Coenzymes do not protect the enzyme from inactivation. Complete maintenance of enzyme activity was achieved in the presence of saturating concentrations of the formate and upon formation of the ternary complex, enzyme-NAD-azide. Formate specifically protects two lysine residues per dimer molecule of the enzyme from modification. The presence of one essential lysine residue in the substrate-binding region of the enzyme active site is assumed.     

Crystal structure of Saccharomyces cerevisiae Aro8, a putative α‐aminoadipate aminotransferase

α‐Aminoadipate aminotransferase (AAA‐AT) catalyzes the amination of 2‐oxoadipate to α‐aminoadipate in the fourth step of the α‐aminoadipate pathway of lysine biosynthesis in fungi. The aromatic aminotransferase Aro8 has recently been identified as an AAA‐AT in Saccharomyces cerevisiae. This enzyme displays broad substrate selectivity, utilizing several amino acids and 2‐oxo acids as substrates. Here we report the 1.91Å resolution crystal structure of Aro8 and compare it to AAA‐AT LysN from Thermus thermophilus and human kynurenine aminotransferase II. Inspection of the active site of Aro8 reveals asymmetric cofactor binding with lysine‐pyridoxal‐5‐phosphate bound within the active site of one subunit in the Aro8 homodimer and pyridoxamine phosphate and a HEPES molecule bound to the other subunit. The HEPES buffer molecule binds within the substrate‐binding site of Aro8, yielding insights into the mechanism by which it recognizes multiple substrates and how this recognition differs from other AAA‐AT/kynurenine aminotransferases.     

Investigation of the conserved lysines of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase

Sequence analysis has revealed two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Crystal structures of ternary complexes of the Class II enzyme from Pseudomonas mevalonii revealed lysine 267 critically positioned at the active site. This observation suggested a revised catalytic mechanism in which lysine 267 facilitates hydride transfer from reduced coenzyme by polarizing the carbonyl group of HMG-CoA and subsequently of bound mevaldehyde, an inference supported by mutagenesis of lysine 267 to aminoethylcysteine. For this mechanism to be general, Class I HMG-CoA reductases ought also to possess an active site lysine. Three lysines are conserved among all Class I HMG-CoA reductases. The three conserved lysines of Syrian hamster HMG-CoA reductase were mutated to alanine. All three mutant enzymes had reduced but detectable activity. Of the three conserved lysines, sequence alignments implicate lysine 734 of the hamster enzyme as the most likely cognate of P. mevalonii lysine 267. Low activity of enzyme K734A did not reflect an altered structure. Substrate recognition was essentially normal, and both circular dichroism spectroscopy and analytical ultracentrifugation implied a native structure. Enzyme K734A also formed an active heterodimer when coexpressed with inactive mutant enzyme D766N. We infer that a lysine is indeed essential for catalysis by the Class I HMG-CoA reductases and that the revised mechanism for catalysis is general for all HMG-CoA reductases.     

A comparison of staphostatin B with standard mechanism serine protease inhibitors

Staphostatins are the endogenous, highly specific inhibitors of staphopains, the major secreted cysteine proteases from Staphylococcus aureus. We have previously shown that staphostatins A and B are competitive, active site-directed inhibitors that span the active site clefts of their target proteases in the same orientation as substrates. We now report the crystal structure of staphostatin B in complex with wild-type staphopain B at 1.9 A resolution. In the complex structure, the catalytic residues are found in exactly the positions that would be expected for uncomplexed papain-type proteases. There is robust, continuous density for the staphostatin B binding loop and no indication for cleavage of the peptide bond that comes closest to the active site cysteine of staphopain B. The carbonyl carbon atom C of this peptide bond is 4.1 A away from the active site cysteine sulfur Sgamma atom. The carbonyl oxygen atom O of this peptide bond points away from the putative oxyanion hole and lies almost on a line from the Sgamma atom to the C atom. The arrangement is strikingly similar to the "ionmolecule" arrangement for the complex of papain-type enzymes with their substrates but differs significantly from the arrangement conventionally assumed for the Michaelis complex of papain-type enzymes with their substrates and also from the arrangement that is crystallographically observed for complexes of standard mechanism inhibitors and their target serine proteases.     

Human prorenin structure sheds light on a novel mechanism of its autoinhibition and on its non-proteolytic activation by the (pro)renin receptor

Antibodies and prorenin mutants have long been used to structurally characterize prorenin, the inactive proenzyme form of renin. They were designed on the basis of homology models built using other aspartyl protease proenzyme structures since no structure was available for prorenin. Here, we present the first X-ray structure of a prorenin. The current structure of prorenin reveals that, in this zymogene, the active site of renin is blocked by the N-terminal residues of the mature version of the renin molecule, which are, in turn, covered by an Ω-shaped prosegment. This prevents access of substrates to the active site. The departure of the prosegment on activation induces an important global conformational change in the mature renin molecule with respect to prorenin: similar to other related enzymes such as pepsin or gastricsin, the segment that constitutes the N-terminal β-strand in renin is displaced from the renin active site by about 180° straight into the position that corresponds to the N-terminal β-strand of the prorenin prosegment. This way, the renin active site will become completely exposed and capable of carrying out its catalytic functions. A unique inactivation mechanism is also revealed, which does not make use of a lysine against the catalytic aspartates, probably in order to facilitate pH-independent activation [e.g., by the (pro)renin receptor].     

The porphobilinogen synthase catalyzed reaction mechanism

Porphobilinogen synthase (PBGS) catalyzes the first common reaction in the biosynthesis of the tetrapyrroles, the asymmetric condensation of two molecules of delta-aminolevulinic acid to form porphobilinogen. There is a variable requirement for an essential active site zinc that necessitates consideration of PBGS as an enzyme that may exhibit phylogenetic diversity in its chemical reaction mechanism. Recent crystal structures suggest reaction mechanisms that involve two covalent Schiff base linkages between adjacent active site lysine residues and each of the two substrate molecules. The reaction appears to stall at a covalently bound almost-product intermediate that is poised for breakdown to product upon binding of a substrate molecule to an adjacent active site and a subsequent conformational change.     

The mechanism of addition of pyridoxal 5'-phosphate to Escherichia coli apo-serine hydroxymethyltransferase

Previous studies suggest that the addition of pyridoxal 5'-phosphate to apo-serine hydroxymethyltransferase from Escherichia coli is the last event in the enzyme's folding process. We propose a mechanism for this reaction based on quenched-flow, stopped-flow and rapid-scanning stopped-flow experiments. All experiments were performed with an excess of apo-enzyme over cofactor, since excess pyridoxal 5'-phosphate results in a second molecule of cofactor binding to Lys346, which is part of the tetrahydropteroylglutamate-binding site. The equilibrium between the aldehyde and hydrate forms of the cofactor affects the kinetics of addition to the active site. Direct evidence of the formation of an intermediate aldimine between the cofactor and the active-site lysine was obtained. The results have been interpreted according to a three-step mechanism in which: (i) both aldehyde and hydrate forms of the cofactor bind rapidly and non-covalently to the apo-enzyme; (ii) only the aldehyde form reacts with the active-site lysine to give an intermediate internal aldimine with unusual spectral properties; and (iii) a final conformational change gives the native holo-enzyme.     

Molecular mechanisms of O-GlcNAcylation

Protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification of serines/threonines on metazoan proteins and occurring with similar time scales, dynamics and stoichiometry as protein phosphorylation. Levels of this modification are regulated by two enzymes-O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Although the biochemistry of these enzymes and functional implications of O-GlcNAc have been studied extensively, until recently the structures and molecular mechanisms of OGT/OGA were not understood. This review covers a body of recent work that has led to an understanding of the structure of OGA, its catalytic mechanism and the development of a plethora of different inhibitors that are finding their use in cell biological studies towards the functional implications of O-GlcNAc. Furthermore, the very recent structure determination of a bacterial OGT orthologue has given the first insights into the contribution of the tetratricopeptide repeats (TPRs) to the active site and the role of some residues in catalysis and substrate binding.     

Chemical and kinetic mechanisms of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

The chemical and kinetic mechanisms of purified aspartate-beta-semialdehyde dehydrogenase from Escherichia coli have been determined. The kinetic mechanism of the enzyme, determined from initial velocity, product and dead end inhibition studies, is a random preferred order sequential mechanism. For the reaction examined in the phosphorylating direction L-aspartate-beta-semialdehyde binds preferentially to the E-NADP-Pi complex, and there is random release of the products L-beta-aspartyl phosphate and NADPH. Substrate inhibition is displayed by both Pi and NADP. Inhibition patterns versus the other substrates suggest that Pi inhibits by binding to the phosphate subsite in the NADP binding site, and the substrate inhibition by NADP results from the formation of a dead end E-beta-aspartyl phosphate-NADP complex. The chemical mechanism of the enzyme has been examined by pH profile and chemical modification studies. The proposed mechanism involves the attack of an active site cysteine sulfhydryl on the carbonyl carbon of aspartate-beta-semialdehyde, with general acid assistance by an enzyme lysine amino group. The resulting thiohemiacetal is oxidized by NADP to a thioester, with subsequent attack by the dianion of enzyme bound phosphate. The collapse of the resulting tetrahedral intermediate leads to the acyl-phosphate product and liberation of the active site cysteine.     

Crystal structure of fosfomycin resistance kinase FomA from Streptomyces wedmorensis

The fosfomycin resistance protein FomA inactivates fosfomycin by phosphorylation of the phosphonate group of the antibiotic in the presence of ATP and Mg(II). We report the crystal structure of FomA from the fosfomycin biosynthetic gene cluster of Streptomyces wedmorensis in complex with diphosphate and in ternary complex with the nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-imido)-triphosphate (AMPPNP), Mg(II), and fosfomycin, at 1.53 and 2.2 angstroms resolution, respectively. The polypeptide exhibits an open alphabetaalpha sandwich fold characteristic for the amino acid kinase family of enzymes. The diphosphate complex shows significant disorder in loops surrounding the active site. As a result, the nucleotide-binding site is wide open. Binding of the substrates is followed by the partial closure of the active site and ordering of the alpha2-helix. Structural comparison with N-acetyl-L-glutamate kinase shows several similarities in the site of phosphoryl transfer: 1) preservation of architecture of the catalytical amino acids of N-acetyl-L-glutamate kinase (Lys9, Lys216, and Asp150 in FomA); 2) good superposition of the phosphate acceptor groups of the substrates, and 3) good superposition of the diphosphate molecule with the beta- and gamma-phosphates of AMPPNP, suggesting that the reaction could proceed by an associative in-line mechanism. However, differences in conformations of the triphosphate moiety of AMPPNP molecules, the long distance (5.1 angstroms) between the phosphate acceptor and donor groups in FomA, and involvement of Lys18 instead of Lys9 in binding with the gamma-phosphate may indicate a different reaction mechanism. The present work identifies the active site residues of FomA responsible for substrate binding and specificity and proposes their roles in catalysis.     

Mechanism of inhibition of yeast squalene synthase by substrate analog inhibitors.

Squalene synthase catalyzes the reductive condensation of two identical substrate molecules, farnesyl diphosphate, to the hydrocarbon squalene via an obligatory intermediate, presqualene pyrophosphate. Since the kinetic mechanism of the transformation is sequential, two substrate binding pockets that recognize the same molecule must exist in the enzyme active site. This raises the possibility of a choice of binding pockets for inhibitors that are designed as substrate or reaction intermediate analogs and thus may provide some information on the mechanism of differentiation of the two identical molecules. In this report, we have investigated the mechanism of inhibition of a series of farnesyl diphosphate analog inhibitors. The inhibitors fall into two categories. One class of compounds binds to free enzyme as well as the enzyme substrate complex, and the binding is refractory to the concentration of the substrate. The second class binds only to the free enzyme, and its binding is significantly modulated by the substrate concentration. Very modest structural changes in the compounds appear to dictate which class of inhibitor any compound may fall into. The significance of these observations with respect to the mechanism of the enzyme are discussed.     

Conformation and polarity of the active site of xylanase I from Thermomonospora sp. as deduced by fluorescent chemoaffinity labeling. Site and significance of a histidine residue.

A fluorescent chemoaffinity label o-phthalaldehyde (OPTA) was used to ascertain the conformational flexibility and polarity at the active site of xylanase I (Xyl I). The kinetics of inactivation of Xyl I with OPTA revealed that complete inactivation occurred due to the binding of one molecule of OPTA to the active site of Xyl I. The formation of a single fluorescent isoindole derivative corroborated these findings. OPTA has been known to form a fluorescent isoindole derivative by crosslinking the proximal thiol and amino groups of cysteine and lysine. The involvement of cysteine in the formation of a Xyl I-isoindole derivative has been negated by fluorometric and chemical modification studies on Xyl I with group-specific reagents and by amino-acid analysis. The kinetic analysis of diethylpyrocarbonate-modified Xyl I established the presence of an essential histidine at or near the catalytic site of Xyl I. Modification of histidine and lysine residues by diethylpyrocarbonate and 2,4,6-trinitrobenzenesulfonic acid, respectively, abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating that histidine and lysine participate in the formation of the isoindole complex. A mechanism for the reaction of OPTA with histidine and lysine residues present in the protein structure has been proposed. Experimental evidence presented here suggests for the first time that the active site of Xyl I is conformationally more flexible and more easily perturbed in the presence of denaturants than the molecule as a whole. The changes in the fluorescence emission maxima of a model compound (isoindole adduct) in solvents of different polarity were compared with the fluorescence behaviour of the Xyl I-isoindole derivative, leading to the conclusion that the active site is located in a microenvironment of low polarity.     

Crystal structure of lysine sulfonamide inhibitor reveals the displacement of the conserved flap water molecule in human immunodeficiency virus type 1 protease

Human immunodeficiency virus type 1 (HIV-1) protease has been continuously evolving and developing resistance to all of the protease inhibitors. This requires the development of new inhibitors that bind to the protease in a novel fashion. Most of the inhibitors that are on the market are peptidomimetics, where a conserved water molecule mediates hydrogen bonding interactions between the inhibitors and the flaps of the protease. Recently a new class of inhibitors, lysine sulfonamides, was developed to combat the resistant variants of HIV protease. Here we report the crystal structure of a lysine sulfonamide. This inhibitor binds to the active site of HIV-1 protease in a novel manner, displacing the conserved water and making extensive hydrogen bonds with every region of the active site.