Enhanced Survival in Mice Exposed to Ionizing Radiation by Combination of Gamma-Tocotrienol and Simvastatin.

Ionizing radiation exposure is a major concern for active military service members, as well as civilian population. Considering that the exposure is not predictable, it is imperative that strategies to counteract radiation damage must be discovered. Recent in vitro studies performed in our laboratory demonstrated that the vitamin E analog gamma-tocotrienol (GT3) in combination with cholesterol-lowering drugs (Statins), synergistically induced endothelial thrombomodulin, an anticoagulant with radio-protective efficacy. It was hypothesized that the combination of treatment with both GT3 along with Statins would provide better radiation protection in vivo than each drug individually. CD2F1 mice were injected subcutaneously with either vehicle or single dose of GT3 (200 mg/kg body weight) 24 hours before irradiation followed by oral or subcutaneous administration of various doses of simvastatin (25, 50, and 100 mg/kg body weight) before exposure to lethal doses (11.5 and 12 Gy) of Cobalt-60 (60Co) gamma-irradiation. The combined treatment group exhibited enhanced radiation lethality protection substantially, accelerated white blood cell recovery, and augmented restoration of bone marrow cellularity when compared to the animals treated with either drug exclusively. This information clearly suggests that combined treatment could be used as a safeguard for military personnel from exposure to harmful ionizing radiation.

Supporting role of lysine 13 and glutamate 16 in the acid-base mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae.

Saccharopine dehydrogenase (SDH) catalyzes the NAD+ dependent oxidative deamination of saccharopine to form lysine (Lys) and α-ketoglutarate (α-kg). The active site of SDH has a number of conserved residues that are believed important to the overall reaction. Lysine 13, positioned near the active site base (K77), forms a hydrogen bond to E78 neutralizing it, and contributing to setting the pKa of the catalytic residues to near neutral pH. Glutamate 16 is within hydrogen bond distance to the Nε atom of R18, which has strong H-bonding interactions with the α-carboxylate and α-oxo groups of α-kg. Mutation of K13 to M and E16 to Q decreased kcat by about 15-fold, and primary and solvent deuterium kinetic isotope effects measured with the mutant enzymes indicate hydride transfer is rate limiting for the overall reaction. The pH-rate profiles for K13M exhibited no pH dependence, consistent with an increase in negative charge in the active site resulting in the perturbation in the pKas of catalytic groups. Elimination of E16 affects optimal positioning of R18, which is involved in binding and holding α-kg in the correct conformation for optimum catalysis. In agreement, a ΔΔG°' of 2.60 kcal/mol is estimated from the change in Kα-kg for replacing E16 with Q.

Evidence in support of lysine 77 and histidine 96 as acid-base catalytic residues in saccharopine dehydrogenase from Saccharomyces cerevisiae.

Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the α-aminoadipate pathway, the conversion of l-saccharopine to l-lysine (Lys) and α-ketoglutarate (α-kg) using NAD⁺ as an oxidant. The enzyme utilizes a general acid-base mechanism to conduct its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and a group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo form and a closed form of the enzyme with saccharopine and NADH bound have been determined at 2.0 and 2.2 Å resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II that closes the active site cleft between the two domains and brings H96 and K77 into the proximity of the substrate binding site is observed. The hydride transfer distance is 3.6 Å, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. Preparation of the K77M and H96Q single-mutant and K77M/H96Q double-mutant enzymes provides data consistent with their role as the general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the ε-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/E(t) and >10³-fold increases in V2/K(Lys)E(t) and V2/K(α-kg)E(t) (the double mutation gives >105-fold decreases in the second-order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V2/K(Lys). A value of 2.0 was also observed for (D)(V2/K(Lys))(D2O) when the primary deuterium kinetic isotope effect was repeated in D2O, consistent with a rate-limiting hydride transfer step. A viscosity effect of 0.8 was observed on V2/K(Lys), indicating the solvent deuterium isotope effect resulted from stabilization of an enzyme form prior to hydride transfer. A small normal solvent isotope effect is observed on V, which decreases slightly when repeated with NADD, consistent with a contribution from product release to rate limitation. In addition, V2/K(Lys)E(t) is pH-independent, which is consistent with the loss of an acid-base catalyst and perturbation of the pK(a) of the second catalytic group to a higher pH, likely a result of a change in the overall charge of the active site. The primary deuterium kinetic isotope effect for H96Q, measured in H2O or D2O, is within error equal to 1. A solvent deuterium isotope effect of 2.4 is observed with NADH or NADD as the dinucleotide substrate. Data suggest rate-limiting imine formation, consistent with the proposed role of H96 in protonating the leaving hydroxyl as the imine is formed. The pH-rate profile for V2/K(Lys)E(t) exhibits the pK(a) for K77, perturbed to a value of ∼9, which must be unprotonated to accept a proton from the ε-amine of the substrate Lys so that it can act as a nucleophile. Overall, data are consistent with a role for K77 acting as the base that accepts a proton from the ε-amine of the substrate lysine prior to nucleophilic attack on the α-oxo group of α-ketoglutarate, and finally donating a proton to the imine nitrogen as it is reduced to give saccharopine. In addition, data indicate a role for H96 acting as a general acid-base catalyst in the formation of the imine between the ε-amine of lysine and the α-oxo group of α-ketoglutarate.

Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase.

A novel class of inhibitors of the enzyme γ-glutamyl transpeptidase (GGT) were evaluated. The analog OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction, but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.

Kinetic and chemical mechanisms of homocitrate synthase from Thermus thermophilus.

The homocitrate synthase from Thermus thermophilus (TtHCS) is a metal-activated enzyme with either Mg(2+) or Mn(2+) capable of serving as the divalent cation. The enzyme exhibits a sequential kinetic mechanism. The mechanism is steady state ordered with α-ketoglutarate (α-Kg) binding prior to acetyl-CoA (AcCoA) with Mn(2+), whereas it is steady state random with Mg(2+), suggesting a difference in the competence of the E·Mn·α-Kg·AcCoA and E·Mg·α-Kg·AcCoA complexes. The mechanism is supported by product and dead-end inhibition studies. The primary isotope effect obtained with deuterioacetylCoA (AcCoA-d(3)) in the presence of Mg(2+) is unity (value 1.0) at low concentrations of AcCoA, whereas it is 2 at high concentrations of AcCoA. Data suggest the presence of a slow conformational change induced by binding of AcCoA that accompanies deprotonation of the methyl group of AcCoA. The solvent kinetic deuterium isotope effect is also unity at low AcCoA, but is 1.7 at high AcCoA, consistent with the proposed slow conformational change. The maximum rate is pH independent with either Mg(2+) or Mn(2+) as the divalent metal ion, whereas V/K(α-Kg) (with Mn(2+)) decreases at low and high pH giving pK values of about 6.5 and 8.0. Lysine is a competitive inhibitor that binds to the active site of TtHCS, and shares some of the same binding determinants as α-Kg. Lysine binding exhibits negative cooperativity, indicating cross-talk between the two monomers of the TtHCS dimer. Data are discussed in terms of the overall mechanism of TtHCS.

The oxidation state of active site thiols determines activity of saccharopine dehydrogenase at low pH.

Saccharopine dehydrogenase catalyzes the NAD-dependent conversion of saccharopine to generate L-lysine and α-ketoglutarate. A disulfide bond between cysteine 205 and cysteine 249, in the vicinity of the dinucleotide-binding site, is observed in structures of the apoenzyme, while a dithiol is observed in a structure with AMP bound, suggesting preferential binding of the dinucleotide to reduced enzyme. Mutation of C205 to S gave increased values of V/E(t) and V/KE(t) at pH 7 compared to wild type. Primary deuterium and solvent deuterium kinetic isotope effects suggest the catalytic pathway, which includes the hydride transfer and hydrolysis steps, contributes more to rate limitation in C205S, but the rates of the two steps relative to one another remain the same. There is a large increase in the rate constants V1/E(t) and V1/K(NAD)Et at pH values below 7 compared to WT. Data indicate the low pH increase in activity results from a decreased sensitivity of the C205S mutant enzyme to the protonation state of an enzyme group with a pK(a) of about 7, likely responsible for a pH-dependent conformational change. Reduction of WT and C205S mutant enzymes with TCEP gives equal activities at pH 6, consistent with the increased activity observed for the C205S mutant enzyme.

The multifaceted pyridoxal 5'-phosphate-dependent O-acetylserine sulfhydrylase.

Cysteine is the final product of the reductive sulfate assimilation pathway in bacteria and plants and serves as the precursor for all sulfur-containing biological compounds, such as methionine, S-adenosyl methionine, iron-sulfur clusters and glutathione. Moreover, in several microorganisms cysteine plays a role as a reducing agent, eventually counteracting host oxidative defense strategies. Cysteine is synthesized by the PLP-dependent O-acetylserine sulfhydrylase, a dimeric enzyme belonging to the fold type II, catalyzing a beta-replacement reaction. In this review, the spectroscopic properties, catalytic mechanism, three-dimensional structure, conformational changes accompanying catalysis, determinants of enzyme stability, role of selected amino acids in catalysis, and the regulation of enzyme activity by ligands and interaction with serine acetyltransferase, the preceding enzyme in the biosynthetic pathway, are described. Given the key biological role played by O-acetylserine sulfhydrylase in bacteria, inhibitors with potential antibiotic activity have been developed. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.

Reaction product affinity regulates activation of human sulfotransferase 1A1 PAP sulfation.

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates the activity of bio-signaling molecules and aids in metabolizing hydroxyl-containing xenobiotics. The sulfuryl donor for the SULT reaction is adenosine 3'-phosphate 5'-phosphosulfate (PAPS), while products are adenosine 3',5'-diphosphate (PAP) and a sulfated alcohol. Human phenol sulfotransferase (SULT1A1) is one of the major detoxifying enzymes for phenolic xenobiotics. The mechanism of SULT1A1-catalyzed sulfation of PAP by pNPS was investigated. PAP was sulfated by para-nitrophenyl sulfate (pNPS) in a concentration-dependent manner. 2-Naphthol inhibited sulfation of PAP, competing with pNPS, while phenol activated the sulfation reaction. At saturating PAP, a ping pong kinetic mechanism is observed with pNPS and phenol as substrates, consistent with phenol intercepting the E-PAPS complex prior to dissociation of PAPS. At high concentrations, phenol competes with pNPS, consistent with formation of the E-PAP-phenol dead-end complex. Data are consistent with the previously reported mechanism for sulfation of 2-naphthol by PAPS, and its activation by pNPS. Overall, data are consistent with release of PAP from E-PAP and PAPS from E-PAPS contributing to rate-limitation in both reaction directions.

Identification of the structural determinants for the stability of substrate and aminoacrylate external Schiff bases in O-acetylserine sulfhydrylase-A.

O-Acetylserine sulfhydrylase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the beta-acetoxy group of O-acetyl-L-serine (OAS) by a thiol to give L-cysteine. Previous studies of the K41A mutant enzyme showed L-methionine bound in an external Schiff base (ESB) linkage to PLP as the enzyme was isolated. The mutant enzyme exists in a closed form, optimizing the orientation of the cofactor PLP and properly positioning active site functional groups for reaction. The trigger for closing the active site upon formation of the ESB is thought to be interaction of the substrate alpha-carboxylate with the substrate-binding loop comprised of T68, S69, G70, and N71, and Q142, which is positioned above the cofactor as one looks into the active site. To probe the contribution of these residues to the active site closing and orientation of PLP in the ESB, T68, S69, N71, and Q142 were changed to alanine. Absorbance, fluorescence, near UV-visible CD, and (31)P NMR spectral studies and pre-steady state kinetic studies were used to characterize the mutant enzymes. All of the mutations affect closure of the active site, but to differing extents. In addition, the site appears to be more hydrophilic given that the ESBs do not exhibit a significant amount of the enolimine tautomer. No buildup of the alpha-aminoacrylate intermediate (AA) is observed for the T68A and Q142A mutant enzymes. However, pyruvate is produced at a rate much greater than that of the wild-type enzyme. Data suggest that T68 and Q142 play a role in stabilizing the AA. Both residues donate a hydrogen bond to one of the carboxylate oxygens of the methionine ESB and may also be responsible for the proper orientation of the ESB to generate the AA. The S69A and N71A mutants exhibit formation of the AA, but the rate constant for its formation from the ESB is decreased by 1 order of magnitude compared to that of the wild type. S69 donates a hydrogen bond to the substrate carboxylate in the ESB, while N71 donates hydrogen bonds to O3' of the cofactor and the carboxylate of the ESB; these side chains may also affect the orientation of the ESB. Data suggest that interaction of intermediates with the substrate-binding loop and Q142 gives a properly aligned Michaelis complex and facilitates the beta-elimination reaction.

Scintillation proximity assay for inhibitors of Escherichia coli MurG and, optionally, MraY.

MurG and MraY, essential enzymes involved in the synthesis of bacterial peptidoglycan, are difficult to assay because the substrates are lipidic and hard to prepare in large quantities. Based on the use of Escherichia coli membranes lacking PBP1b, we report a high-throughput method to measure the activity of MurG and, optionally, MraY as well. In these membranes, incubation with the two peptidoglycan sugar precursors results in accumulation of lipid II rather than the peptidoglycan produced by wild-type membranes. MurG was assayed by addition of UDP-[3H]N-acetylglucosamine to membranes in which lipid I was preformed by incubation with UDP-N-acetyl-muramylpentapeptide, and the product was captured by wheat germ agglutinin scintillation proximity assay beads. In a modification of the assay, the activity of MraY was coupled to that of MurG by addition of both sugar precursors together in a single step. This allows simultaneous detection of inhibitors of either enzyme. Both assays could be performed using wild-type membranes by addition of the transglycosylase inhibitor moenomycin. Nisin and vancomycin inhibited the MurG reaction; the MraY-MurG assay was inhibited by tunicamycin as well. Inhibitors of other enzymes of peptidoglycan synthesis--penicillin G, moenomycin, and bacitracin--had no effect. Surprisingly, however, the beta-lactam cephalosporin C inhibited both the MurG and MraY-MurG assays, indicating a secondary mechanism by which this drug inhibits bacterial growth. In addition, it inhibited NADH dehydrogenase in membranes, a hitherto-unreported activity. These assays can be used to screen for novel antibacterial agents.