Biosynthesis of 2-aceto-2-hydroxy acids: acetolactate synthases and acetohydroxyacid synthases.

Two groups of enzymes are classified as acetolactate synthase (EC 4. 1.3.18). This review deals chiefly with the FAD-dependent, biosynthetic enzymes which readily catalyze the formation of acetohydroxybutyrate from pyruvate and 2-oxobutyrate, as well as of acetolactate from two molecules of pyruvate (the ALS/AHAS group). These enzymes are generally susceptible to inhibition by one or more of the branched-chain amino acids which are ultimate products of the acetohydroxyacids, as well as by several classes of herbicides (sulfonylureas, imidazolinones and others). Some ALS/AHASs also catalyze the (non-physiological) oxidative decarboxylation of pyruvate, leading to peracetic acid; the possible relationship of this process to oxygen toxicity is considered. The bacterial ALS/AHAS which have been well characterized consist of catalytic subunits (around 60 kDa) and smaller regulatory subunits in an alpha2beta2 structure. In the case of Escherichia coli isozyme III, assembly and dissociation of the holoenzyme has been studied. The quaternary structure of the eukaryotic enzymes is less clear and in plants and yeast only catalytic polypeptides (homologous to those of bacteria) have been clearly identified. The presence of regulatory polypeptides in these organisms cannot be ruled out, however, and genes which encode putative ALS/AHAS regulatory subunits have been identified in some cases. A consensus sequence can be constructed from the 21 sequences which have been shown experimentally to represent ALS/AHAS catalytic polypeptides. Many other sequences fit this consensus, but some genes identified as putative 'acetolactate synthase genes' are almost certainly not ALS/AHAS. The solution of the crystal structures of several thiamin diphosphate (ThDP)-dependent enzymes which are homologous to ALS/AHAS, together with the availability of many amino acid sequences for the latter enzymes, has made it possible for two laboratories to propose similar, reasonable models for a dimer of catalytic subunits of an ALS/AHAS. A number of characteristics of these enzymes can now be better understood on the basis of such models: the nature of the herbicide binding site, the structural role of FAD and the binding of ThDP-Mg2+. The models are also guides for experimental testing of ideas concerning structure-function relationships in these enzymes, e.g. the nature of the substrate recognition site. Among the important remaining questions is how the enzyme suppresses alternative reactions of the intrinsically reactive hydroxyethylThDP enamine formed by the decarboxylation of the first substrate molecule and specifically promotes its condensation with 2-oxobutyrate or pyruvate.

Herbicidal Activity of an Isopropylmalate Dehydrogenase Inhibitor.

Isopropylmalate dehydrogenase (IPMDH) is the third enzyme specific to leucine biosynthesis. It catalyzes the oxidative decarboxylation of 3-isopropylmalate (3-IPM) to 2-ketoisocaproic acid. The partially purified enzyme from pea (Pisum sativum L.) shows a broad pH optimum of 7.8 to 9.1 and has Km values for 3-IPM and NAD of 18 and 40 [mu]M, respectively. O-Isobutenyl oxalylhydroxamate (O-IbOHA) has been discovered to be an excellent inhibitor of the pea IPMDH, with an apparent inhibitor constant of 5 nM. As an herbicide, O-IbOHA showed only moderate activity on a variety of broadleaf and grass species. We characterized the herbicidal activity of O-IbOHA on corn (Zea mays L.), a sensitive species; giant foxtail (Setaria faberi) and morning glory (Ipomoea purpurea [L.] Roth), moderately tolerant species; and soybean [Glycine max L. Merr.), a tolerant species. Differences in tolerance among the species were not due to differences in the sensitivity of IPMDH. Studies with [14C]O-IbOHA suggested that uptake and translocation were not major limitations for herbicidal activity, nor were they determinants of tolerance. Moreover, metabolism could not account for the difference in tolerance of corn, foxtail, and morning glory, although it might account for the tolerance of soybean. Herbicidal activity on all four species was correlated with the accumulation of 3-IPM in the plants.

High-level synthesis of recombinant HIV-1 protease and the recovery of active enzyme from inclusion bodies.

A complete chemical synthesis and assembly of genes for the production of human immunodeficiency virus type-I protease (HIV-PR) and its precursors are described. The T7 expression system was used to produce high levels of active HIV-PR and its precursors in Escherichia coli inclusion bodies. The gene encoding the open reading frames of HIV-PR was expressed in E. coli as a 10-kDa protein, while the genes encoding HIV-PR precursors were expressed as larger proteins, which were partially processed in E. coli to the 10-kDa form. These processing events are autoproteolytic, since a single-base mutation, changing the active-site aspartic acid to glycine, completely abolished the conversion. HIV-PR can be released with 8 M urea from washed cellular inclusion bodies, resulting in a preparation with few bacterial host proteins. After refolding, this preparation contains no nonspecific protease or peptidase activities. The recombinant HIV-PR isolated from inclusion bodies cleaves HIV-PR substrates specifically with a specific activity comparable to column-purified HIV-PR.

Quo vadis photorespiration: a tale of two aldolases.

An O2-consuming side reaction of D-ribulose 1,5-bisphosphate carboxylase causes photorespiration in plants. This reaction may be an inevitable consequence of the enzyme's inability to protect its ene-diolate reaction intermediate from O2, a notion that is supported by the failure of persistent efforts to eliminate selectively its oxygenase activity by genetic manipulation. We have examined two a1dolases with similar ene-diolate intermediates, L-rhamnulose 1-phosphate aldolase and L-fuculose 1-phosphate aldolase. The former enzyme has an oxygenase activity, while the latter does not, suggesting that the reaction with O2 is not inevitable.

Glutathione carbamoylation with S-methyl N,N-diethylthiolcarbamate sulfoxide and sulfone. Mitochondrial low Km aldehyde dehydrogenase inhibition and implications for its alcohol-deterrent action.

S-Methyl N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO) and sulfone (DETC-MeSO2) both inhibit rat liver low Km aldehyde dehydrogenase (ALDH2) in vitro and in vivo (Nagendra et al., Biochem Pharmacol 47: 1465-1467, 1994). DETC-MeSO has been shown to be a metabolite of disulfiram, but DETC-MeSO2 has not. Studies were carried out to further investigate the inhibition of ALDH2 by DETC-MeSO and DETC-MeSO2. In an in vitro system containing hydrogen peroxide and horseradish peroxidase, the rate of DETC-MeSO oxidation corresponded to the rate of DETC-MeSO2 formation. Carbamoylation of GSH by both DETC-MeSO and DETC-MeSO2 was observed in a rat liver S9 fraction. Carbamoylation of GSH was not observed in the presence of N-methylmaleimide. In in vitro studies, DETC-MeSO and DETC-MeSO2 were equipotent ALDH2 inhibitors when solubilized mitochondria were used, but DETC-MeSO was approximately four times more potent than DETC-MeSO2 in intact mitochondria. In studies with rats, the dose (i.p. or oral) required to inhibit 50% ALDH2 (ED50) was 3.5 mg/kg for DETC-MeSO and approximately 35 mg/kg for DETC-MeSO2, approximately a 10-fold difference. Furthermore, maximum ALDH2 inhibition occurred 1 hr after DET(-MeSO administration, whereas maximal ALDH2 inhibition occurred 8 hr after DETC-MeSO2 dosing. DETC-MeSO is, therefore, not only a more potent ALDH2 inhibitor than DETC-MeSO2 in vivo, but also in vitro when intact mitochondria are utilized. The in vitro results thus support the in vivo findings. Since oxidation of DETC-MeSO can occur both enzymatically and non-enzymatically, it is possible that DETC-MeSO2 is formed in vivo. DETC-MeSO2, however, is not as effective as DETC-MeSO in inhibiting ALDH2, probably because it has difficulty penetrating the mitochondrial membrane. Thus, even if DETC-MeSO2 is formed in vivo from DETC-MeSO, it is the metabolite DETC-MeSO that is most likely responsible for the inhibition of ALDH2 after disulfiram administration.

Studies of NMDA- and non-NMDA-mediated neurotoxicity in cultured neurons.

The neurotoxic effects of various glutamate agonists were studied using whole fetal rat brain cultures. The results showed that L-glutamate (L-glu) and N-methyl-D-aspartate (NMDA) were the most potent agonists for inducing neurotoxicity, producing significant toxicity at 0.10 and 0.01 mM concentrations, respectively. Kainic acid (KA) and quisqualic acid (QA) also produced neurotoxicity, but only at a relatively high concentration (1.0 mM). No other glutamate agonist tested produced neurotoxicity in the cultures following brief incubations. The effects of each agonist were found to be Ca2+ dependent, and the selective NMDA Ca2+ channel agonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine hydrogen maleate (MK-801), blocked the toxicity produced by all the glutamate agonists. Thus, the results of this study found little or no evidence for a direct non-NMDA receptor mediated neurotoxicity. These results suggest that the neurotoxicity produced by the non-NMDA agonists may be due to one of the following mechanisms: (i) non-specific binding of non-NMDA agonists to NMDA receptor; (ii) release of L-glu via non-NMDA agonists induced depolarization of cell membrane and subsequent activation of NMDA receptor by released L-glu; (iii) inhibition of L-glu uptake by non-NMDA agonists resulting in activation of L-glu receptors including NMDA receptors.

New targets for the treatment of vascular diseases and diabetes.

This symposium was organized by Dan Flynn (Monsanto Life Sciences, USA) and Timothy M Willson (Glaxo Wellcome, USA). PPARs (peroxisome proliferator-activated receptors), are nuclear hormone receptors that govern glucose and lipid homeostasis. There are several subtypes of receptors that share activation by unsaturated fatty acids and work in combination with retinoic acid receptors (RXR), which were a topic covered in an earlier symposium. Two classes of chemistry were discussed at the symposium: (i) thiazolidinediones, that interact directly with PPARs and alter lipid metabolism; and, (ii) benzothiepines or benzothiazepines, that inhibit the ileal bile acid transporter (IBAT) and reduce cholesterol levels by increasing bile acid excretion.

Abeles symposium.

This symposium was the inspiration of Joanne Stubbe (MIT, MA, USA), who organized an impressive tribute to the career of Robert H Abeles (Brandeis University, MA, USA). Professor Abeles is an influential figures in the development of 'rational drug design' and is one of the pioneers in the development of suicide substrates. His work in transition-state or reaction-intermediate analogs, most notably trifluoromethyl ketones, has had a substantial impact on the design of novel pharmaceutical compounds. Despite retirement and failing health, Professor Abeles has managed to contribute to the development of combinatorial chemistry, which is currently of interest as a strategy in drug design. Another facet of his career has been a fascination with biosynthetic pathways or enzyme catalyzed reactions of an unusual or obscure nature. Professor Abeles's approach has always had a distinctly chemical emphasis resulting in unraveling detailed, and often surprising, mechanisms. He has served as an inspiration to countless academic and industrial scientists around the world. Evidence of this is the impressive attendance at the Abeles Symposium and the standing ovation given to Professor Abeles. Former associates gave most of the oral presen-tations in the morning and afternoon sessions of the symposium. Professor Joanne Stubbe gave the introductory talk for the morning's session. An overview of the various research areas by Professor Abeles, including his early work on ribonucleotide reductase that led, in part, to Professor Stubbe's own research in this area, was presented.

Regulation of taurine biosynthesis and its physiological significance in the brain.

Cysteine sulfinic acid decarboxylase (CSAD), the rate-limiting enzyme in taurine biosynthesis, was found to be activated under conditions that favor protein phosphorylation and inactivated under conditions favoring protein dephosphorylation. Direct incorporation of 32P into purified CSAD has been demonstrated with [gamma 32P]ATP and PKC, but not PKA. In addition, the 32P labeling of CSAD was inhibited by PKC inhibitors suggesting that PKC is responsible for phosphorylation of CSAD in the brain. Okadaic acid had no effect on CSAD activity at 10 microM suggesting that protein phosphatase-2C (PrP-2C) might be involved in the dephosphorylation of CSAD. Furthermore, it was found that either glutamate- or high K(+)-induced depolarization increased CSAD activity as well as 32P-incorporation into CSAD in neuronal cultures, supporting the notion that the CSAD activity is endogenously regulated by protein phosphorylation in the brain. A model to link neuronal excitation, phosphorylation of CSAD and increase in taurine biosynthesis is proposed.

Protein crystal growth aboard the U.S. space shuttle flights STS-31 and STS-32.

NASA: The first microgravity protein crystal growth experiments were performed on Spacelab I by Littke and John. These experiments indicated that the space grown crystals, which were obtained using a liquid-liquid diffusion system, were larger than crystals obtained by the same experimental system on earth. Subsequent experiments were performed by other investigators on a series of space shuttle missions from 1985 through 1990. The results from two of these shuttle flights (STS-26 and STS-29) have been described previously. The results from these missions indicated that the microgravity grown crystals for a number of different proteins were larger, displayed more uniform morphologies, and yielded diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth. This paper presents the results obtained from shuttle flight STS-32 (flown in January, 1990) and preliminary results from the most recent shuttle flight, STS-31 (flown in April, 1990).^ieng

Comparative affinities of the epimeric reaction-intermediate analogs 2- and 4-carboxy-D-arabinitol 1,5-bisphosphate for spinach ribulose 1,5-bisphosphate carboxylase.

2-Carboxy-3-keto-D-arabinitol 1,5-bisphosphate is a tightly bound intermediate of the carboxylase reaction of ribulosebisphosphate carboxylase/oxygenase. Two stereoisomers of an analog of this intermediate, 2-carboxy-D-arabinitol 1,5-bisphosphate (2CABP) and 4-carboxy-D-arabinitol 1,5-bisphosphate (4CABP), are exceptionally potent, virtually irreversible inhibitors of the spinach carboxylase, presumably due to their structural similarity to the gem-diol (hydrated carbonyl at C-3) form of the intermediate. Incubation of the enzyme with either leads to time-dependent loss of activity. Inhibition of the enzyme is biphasic, with initial dissociation constants of 0.47 and 0.19 microM and maximal rates for tight complex formation of 2.2 and 1.8 min-1 for 2CABP and 4CABP, respectively. These values give second-order rate constants for tight complex formation of 7.8 x 10(4) and 1.6 x 10(5) M-1 s-1. To determine the overall affinity of the spinach enzyme for 2CABP and 4CABP, the release rates were determined by dual isotope exchange (3H-inhibitor complex with free 14C-inhibitor). Exchange half-times of 1.82 and 530 days were observed for 4CABP and 2CABP, respectively. Overall dissociation constants of 28 pM (2.8 x 10(-11) M) and 190 fM (1.9 x 10(-13) M) were calculated from these dissociation rates together with the rates of association determined by inactivation kinetics. The difference in affinity of 2CABP and 4CABP corresponds to 2.9 kcal/mol, presumably reflecting the difference in interaction of the enzyme with the two hydroxyls of the intermediate's gem-diol. The kinetic behavior of these two inhibitors, in particular the rather slow maximal rates of association, are consistent with the expected behavior of analogs of a labile intermediate of an enzymic reaction that is far more stable than a transition state.

The sulfonylurea herbicide sulfometuron methyl is an extremely potent and selective inhibitor of acetolactate synthase in Salmonella typhimurium.

The sulfonylurea herbicide sulfometuron methyl inhibits the growth of several bacterial species. In the presence of L-valine, sulfometuron methyl inhibits Salmonella typhimurium, this inhibition can be reversed by L-isoleucine. Reversal of growth retardation by L-isoleucine, accumulation of guanosine 5'-diphosphate 3'-diphosphate (magic spot), and relA mutant hypersensitivity suggest sulfometuron methyl interference with branched-chain amino acid biosynthesis. Growth inhibition of S. typhimurium is mediated by sulfometuron methyl's inhibition of acetolactate synthase, the first common enzyme in the branched-chain amino acid biosynthetic pathway. Sulfometuron methyl exhibits slow-binding inhibition of acetolactate synthase isozyme II from S. typhimurium with an initial Ki of 660 +/- 60 nM and a final, steady-state Ki of 65 +/- 25 nM. Inhibition of acetolactate synthase by sulfometuron methyl is substantially more rapid (10 times) in the presence of pyruvate with a maximal first-order rate constant for conversion from initial to final steady-state inhibition of 0.25 +/- 0.07 min-1 (minimal half-time of 2.8 min). Mutants of S. typhimurium able to grow in the presence of sulfometuron methyl were obtained. They have acetolactate synthase activity that is insensitive to sulfometuron methyl because of mutations in or near ilvG, the structural gene for acetolactate synthase isozyme II.

Oxygenase side reactions of acetolactate synthase and other carbanion-forming enzymes.

Enzymes that mediate carbanion chemistry must protect their reactants from solvent and undesirable electrophiles, such as molecular oxygen. A number of enzymes that utilize carbanionic intermediates were surveyed for O2-consuming side reactions. Several of these enzymes, acetolactate synthase, pyruvate decarboxylase, class II aldolase, and glutamate decarboxylase, catalyze previously undetected oxygen-consuming reactions, while others such as class I aldolase, [(phosphoribosyl)amino]imidazole carboxylase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, and triosephosphate isomerase do not. Prior to this work, only ribulosebisphosphate carboxylase was known to catalyze an oxygenase side reaction. These new example indicate that while O2-consuming side reactions are a more general feature of enzyme-mediated carbanion chemistry than has been previously appreciated, they are not necessarily an inevitable consequence of this chemistry. Expression of an oxygenase activity not only depends on the accessibility of the carbanionic intermediate to molecular oxygen but also may depend on the ability of the enzyme to stabilize the initially formed peroxide anion either through protonation with an appropriate enzymic group or through metal coordination.

Enzyme-coupled assays for proteases.

We have developed a general strategy for assaying proteases that does not require the use of fluorogenic, chromogenic, or radiolabeled peptide substrates. The endo- or exoproteolytic hydrolysis of simple peptides can be followed spectrophotometrically by coupling the proteolytic event via enzyme-catalyzed reactions to a chromogenic redox dye. The couple can be used directly to follow the action of carboxy or amino peptidases on peptide substrates or can be coupled by use of carboxy or amino peptidases to follow the action of endoproteases on peptide substrates that are blocked at the amino or carboxy terminus, respectively. Liberated amino acids are detected by use of amino acid oxidase, oxygen, horseradish peroxidase, and the redox dye 2,2'-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid (epsilon 414nm = 36,000 M-1 cm-1).

Oxalyl hydroxamates as reaction-intermediate analogues for ketol-acid reductoisomerase.

N-Hydroxy-N-isopropyloxamate (IpOHA) is an exceptionally potent inhibitor of the Escherichia coli ketol-acid reductoisomerase. In the presence of Mg2+ or Mn2+, IpOHA inhibits the enzyme in a time-dependent manner, forming a nearly irreversible complex. Nucleotide, which is essential for catalysis, greatly enhances the binding of IpOHA by the reductoisomerase, with NADPH (normally present during the enzyme's rearrangement step, i.e., conversion of a beta-keto acid into an alpha-keto acid, in either the forward or reverse physiological reactions) being more effective than NADP. In the presence of Mg2+ and NADPH, IpOHA appears to bind to the enzyme in a two-step mechanism, with an initial inhibition constant of 160 nM and a maximum rate of formation of the tight, slowly reversible complex of 0.57 min-1 (values that give an association rate of IpOHA, at low concentration, of 5.9 X 10(4) M-1 s-1). The rate of exchange of [14C]IpOHA from an enzyme-[14C]IpOHA-Mg2(+)-NADPH complex with exogenous, unlabeled IpOHA has a half-time of 6 days (150 h). This dissociation rate (1.3 X 10(-6) s-1) and the association rate determined by inactivation kinetics define an overall dissociation constant of 22 pM. By contrast, in the presence of Mn2+ and NADPH, the corresponding association and dissociation rates for IpOHA are 8.2 X 10(4) M-1 s-1 and 3.2 X 10(-6) s-1 (half-time = 2.5 days), respectively, which define an overall dissociation constant of 38 pM. In the presence of NADP or in the absence of nucleotide (both in the presence of Mg2+), the enzyme-IpOHA complex is far more labile, with dissociation half-times of 28 and 2 h, respectively. In the absence of Mg2+ or Mn2+, IpOHA does not exhibit time-dependent inhibition of the reductoisomerase.(ABSTRACT TRUNCATED AT 250 WORDS)