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The substrate specificity of the yeast Silent information regulator 2 (Sir2), the yeast HST2 and the human SIRT2 homologues toward various monoacetylated histone H3 and H4 peptides are analysed to study the basic kinetic mechanism and individual chemical steps of the Sir2 reaction. Steady-state kinetic analysis reveals that ySir2, HST2 and SIRT2 exhibit varying catalytic efficiencies and display a preference among the monoacetylated peptide substrates.

The use of kinetic isotope effect (KIE) measurements to probe the transition-state structure for phosphoryl transfer from meta-nitrobenzyl phosphate(mNBP) to the dual-specificity phosphatase (DSP) Vaccinia H1-related (VHR) is described. An enzymatic mechanism that employs the dianionic form of mNBP as the substrate is suggested where a proton donated by the acid (ASP-92 in VHR) is transferred to the ester oxygen.

Research has been conducted on adenine phosphoribosyltransferase which catalyzes adenine phosphoribosylation from alpha-D-5-phosphoribosyl-1-pyrophosphate. The structural basis for the catalysis has been investigated and the results are reported.

The crystal structure of the inactive human VHR protein phosphatase mutant in complex with the bisphosphorylated MAP kinase peptide reveals that the two phospho residues are situated in distinct pockets and the phosphotyrosine binds to the exposed active site cleft formed by the residues.

Research examines the effect of the activator extracellular regulated kinase (ERK) on the transition state of the phosphoryl transfer reaction mediated by phosphatase MP3. Data show that Asp-262 is a general acid catalyst of ERK-dependent and -independent catalytic activation as well as a general base for the thiol-phosphate intermediate hydrolysis.

The acetylation of histones by human transcriptional coactivator P/CAF proceeds via a fully ordered Bi-Bi kinetic mechanism and chemical catalysis is the rate-limiting step. This enzyme belongs to the GNAT superfamily of N-acetyltransferases.

Three protein tyrosine phosphatases PTPs, namely vaccinia H1-related, leukocyte antigen-related and PTP1, were studied to determine the feasibility of using PTPs as target of transient production of hydrogen peroxide. The results showed that PTPs rapidly react even with very small concentrations of hydrogen peroxide to oxidize the important catalytic cysteine residue to a more stable sulfenic acid intermediate. On the other hand, negligible effect was noted for lambda phosphatase, calcineurin and serine/threonine protein phosphatases PP2C-alpha.

The role of the Asp-181 residue of during the formation of phospho-enzyme intermediate from the mammalian protein-tyrosine-phosphatase (PTP1) was analyzed by site-directed mutagenesis. Site-directed mutagenesis of the Asp-181 and Ser-222 of PTP1 induced minute alterations in the tertiary structure of the enzyme. However, the presteady state reaction of the S22A mutant exhibited a burst of p-nitrophenol formation followed by a slow linear rate due to a rate-limiting intermediate hydrolysis.

The transition state for phosphoryl transfer in human dual-specific phosphatase VHR with p-nitrophenyl phosphate was investigated using kinetic isotope effect measurements. Data were compared with kinetic isotope effects previously found for D92N and S131A mutants. The results revealed similar transition-state structure for catalyzed and uncatalyzed reactions and a resemblance to those for protein-tyrosine phosphatases YOP51 and PTP1. Changes in kinetic isotope effects were attributed to electrostatic effects.

The low molecular weight protein tyrosine phosphatase (low Mr PTPase), Stp1, produced by the yeast Schizosaccharomyces pombe, acts as a phosphatase to aryl and alkyl phosphates and dephosphorylatess phosphotyrosyl peptides and proteins. The amino acid sequence of Stp1 is similar to that of mammalian low Mr PTPase but its activity is almost six times slower. The decomposition of the enzyme intermediate is the rate limiting step in the reaction. Low Mr PTPase are probably dual specificity enzymes as bovine low Mr PTPase removes the phosphate from phosphotyrosyl and phosphoseryl/threonyl proteins.

Enzyme assay reveals that compared to native human dual-specific protein-tyrosine, phosphatase, the aspartic acid-92-aspargine (D92N) mutant enzyme exhibits 100-fold less activity. Presence of protonated native enzyme D92 is necessary for catalytic activity. In the rate limiting step, the D92 residue protonates the phenolate ion by functioning as a general acid. D92 facilitates the hydrolysis of the phosphoenzyme intermediate by acting as a general base.

The intrinsic primary, secondary and solvent kinetic isotope effects of D-amino acid oxidase that catalyzes the D-amino acid oxidation show the inconsistency of the CH bond cleavage with concerted mechanisms. The isotopic effects are evaluate using three substrates of D-serine, D-alanine and glycine. The oxidation reaction of the D-amino acids involves the transfers of the electrons to molecular oxygen.

The effects of hydronium ion concentration and deuterium kinetic isotope on D-amino acid oxidase reductive half-reaction was investigated using different amino acid substrates in rapid or steady state reactions. Results from the detailed studies undertaken indicate that when enzyme concentration is elevated, D-serine is slightly sticky owing to the depression of the substrate dissociation rate. D-alanine reacts very slowly with the enzyme while D-glycine reacts maximally at basic pH.

The first 21 residues of histone H4 were used to design a postranslational modification-randomized, combinatorial library to systematically examine the proteins that interpret a histone code. The data obtained from the one-bead, one-compound (OBOC) histone tail libraries could be applied to probe the specificity of histone modifying enzymes and histone-binding protein.

The article explains the mechanism of the inhibition of silent information regulator 2 (Sir2) protein deacetylases by using thioacetyl-lysine peptide. The peptide is found to exhibit excellent inhibition of the Sir2 deacetylases.

A variety of biochemical approaches are used to compare histone-based substrates of increasing complexity to determine the critical components of nucleosome recognition by the MOZ, Ybf2/Sas3, Sas2, Tip60 family HAT complex, Piccolo NuA4. The histone-fold domain (HFD) regions of histones, particularly residues 21-52 of H4 are discovered to be critical for tight binding and efficient tail acetylation.

Distinct catalytic mechanisms are proposed for the Gcn5 and MYST histone acetyltransferase (HAT) families. The analysis has suggested that MYST family HATs has used a direct-attack mechanism within an Esa1.acetyl-CoA.histone ternary complex, which is similar to that established for the Gcn5 HAT family.

X-ray crystal and NMR solution structures of the protein coded for by Arabidopsis thaliana gene Atlg77540 are described. The structures confirm that Atlg77540 is a member of GNAT superfamily and the structures exhibit structural similarity to members of the GNC5-related N-acetyltransferase family of enzymes.

A direct evidence supporting the nucleophilic attack of the acetyllysine oxygen at the alpha face of the nicotinamide ribose to form a covalent alpha-1'-alkylamidate intermediate and also a biochemical evidence for the role of the active site histidine as a general base and for the identity of the rate-limiting step in the Sir2 catalysis are reported. The implications of the evidence on the mechanisms of deacetylation and possible ADP-ribosylation catalyzed by Sir2 deacetylases are discussed.

A one-bead, one-compound (OBOC) acetyl-peptide library with a quantum dot tagging and fluorescent bead-sorting instrumentation is utilized to develop a novel, high-throughput method for determining deacetylase substrate specificity. In-solution deacetylase assays on randomly chosen hit and nonhit sequences have shown that hits correlated with increased catalytic activity by as much as 20-fold and SIRT1 can discriminate peptide substrates in a context-dependent fashion.

The authiors have investigated human serine/threonine phosphatase PP2C(alpha). The results of X-ray structural analysis have indicated that PP2C(alpha) has an active site containing a dinuclear metal ion center coordinated by invariant carboxylate residues.

Sir2 protein deacetylases (or sirtuins) catalyze NAD+-dependent conversion of ϵ-amino- acetylated lysine residues to deacetylated lysine, nicotinamide, and 2′-O-acetyl-ADP-ribose. Small-molecule modulation of sirtuin activity might treat age-associated diseases, such as type II diabetes, obesity, and neurodegenerative disorders. Here, we have evaluated the mechanisms of sirtuin inhibition of histone peptides containing thioacetyl or mono–, di–, and trifluoroacetyl groups at the ϵ-amino of lysine. Although all substituted peptides yielded inhibition of the deacetylation reaction, the thioacetyl-lysine peptide exhibited exceptionally potent inhibition of sirtuins Sirt1, Sirt2, Sirt3, and Hst2. Using Hst2 as a representative sirtuin, the trifluoroacetyl-lysine peptide displayed competitive inhibition with acetyl-lysine substrate and yielded an inhibition constant (Kis) of 4.8 μM similar to its Kd value of 3.3 μM. In contrast, inhibition by thioacetyl-lysine peptide yielded an inhibition constant (Kis) of 0.0 17 μM, 280-fold lower than its Kd value of 4.7 μM. Examination of thioacetyl-lysine peptide as an alternative sirtuin substrate revealed conserved production of deacetylated peptide and 1′-SH-2′-O-acetyl-ADP-ribose. Pre-steady-state and steady-state analysis of the thioacetyl-lysine peptide showed rapid nicotinamide formation (4.5 s-1) but slow overall turnover (0.0024 s-1), indicating that the reaction stalled at an intermediate after nicotinamide formation. Mass spectral analysis yielded a novel species (m/z 1754.3) that is consistent with an ADP- ribose-peptidyl adduct (1′-S-alkylamidate) as the stalled intermediate. Additional experiments involving solvent isotope effects, general base mutational analysis, and density functional calculations are consistent with impaired 2′-hydroxyl attack on the ADP-ribose-peptidyl intermediate. These results have implications for the development of mechanism-based inhibitors of Sir2 deacetylases. [ABSTRACT FROM AUTHOR]