Oxathiin carboxanilide, a potent inhibitor of human immunodeficiency virus reproduction.

Oxathiin carboxanilide (OC), NSC 615985, a compound originally synthesized as a potential fungicide, was demonstrated to be highly active in preventing human immunodeficiency virus (HIV)-induced cell killing and in inhibiting HIV reproduction. Virus-infected CD4+ lymphocytes were completely protected by 0.5 microM OC, whereas no toxicity was observed at concentrations below 50 microM OC. Production of infectious virus, viral p24 antigen, and virion reverse transcriptase were reduced by OC at concentrations that prevented viral cell killing. A variety of CD4+ T-cell lines were protected by OC from HIV cytopathicity, and OC inhibited two distinct strains of HIV-1. However, HIV-2 infections were unaffected by OC. OC had no direct effect on virions of HIV or on the enzymatic activities of HIV reverse transcriptase or HIV protease. Time-limited treatments of cells with OC before, during, or after exposure of cells to virus failed to protect cells from the eventual cytopathic effects of HIV, and OC failed to inhibit the production of virus from cells in which infection was established or from chronically infected cells. We conclude that the highly active OC has a reversible effect on some early stage of HIV-1 reproduction and cytopathicity. Pilot in vivo experiments showed that circulating concentrations of OC exceeding 1 microM could be achieved and sustained in hamsters for at least a week with no remarkable toxicological sequelae. OC represents a new class of anti-HIV agents that are promising candidates for drug development.

Multiple forms of ubiquitin-protein ligase. Binding of activated ubiquitin to protein substrates.

Enzyme activities that catalyzed the covalent attachment of ubiquitin to protein substrates (ubiquitin-protein ligase, UbL) were purified from the extracts of human red blood cells. These activities required the presence of ubiquitin-activating enzyme and ATP for activity. Four fractions (UbL A, B1, B2, and C) were resolved and showed different specificities toward added substrates [carboxymethylated bovine serum albumin (CM-BSA), G-actin, lysozyme, and alpha-lactalbumin]. The enzyme fractions gave different products with a given substrate. UbL A and UbL B1 were exclusively active with CM-BSA and alpha-lactalbumin, respectively. UbL B2 was most active toward CM-BSA with substantial activities to G-actin and alpha-lactalbumin and with no activity to lysozyme. UbL C showed significant activities with all four substrates, having a highest activity toward CM-BSA. There were many endogenous proteins present in the erythrocyte extract which were efficient substrates for ubiquitin conjugation. In particular, a pair of substrates were identified from erythrocyte extracts which were far more efficient substrates than the denatured proteins exogenously added.

Heterogeneity in the actin activation of myosin.

The soluble proteolytic fragments of myosin, heavy meromyosin and subfragment 1, were prepared with varying amounts of the proteases chymotrypsin and papain, respectively. The actin-activated ATP hydrolysis were examined with oxygen-18-labeled ATP. Each preparation of heavy meromyosin and subfragments 1 displayed two pathways of ATP hydrolysis, called respectively the high and low oxygen exchange mechanisms. The contributions of the two mechanisms were found to be sensitive to the potassium chloride concentration. With a fixed concentration of actin (300 microM), the contribution of the low-exchange mechanism decreased from a maximum of 90% of the ATP hydrolysis at 10 and 20 mM KCl to 12% at 180 mM KCl. The results suggested that the two mechanisms were competing reactions catalyzed by a single species of myosin.

Gamma-butyrobetaine hydroxylase: stereochemical course of the hydroxylation reaction.

The stereochemical course of the aliphatic hydroxylation of gamma-butyrobetaine by calf liver and by Pseudomonas sp AK1 gamma-butyrobetaine hydroxylases has been determined. With [3(RS)-3-3H]-gamma-butyrobetaine or [3(R)-3-3H]-gamma-butyrobetaine as substrate, a rapid and significant loss of tritium to the medium occurred. On the other hand, with [3(S)-3-3H]-gamma-butyrobetaine, only a negligible release of tritium to the aqueous medium was observed. Indeed, on hydroxylation of [3(S)-3-2H]-gamma-butyrobetaine by either the calf liver or bacterial hydroxylase, the isolated product L-carnitine was found to have retained all of the deuterium initially present in the 3(S) position. Since the absolute configuration of the product L-carnitine has been determined to be R, such results are only compatible with a hydroxylation reaction that proceeded with retention of configuration. With [methyl-14C,3(R)-3-3H]-gamma-butyrobetaine as substrate for the calf liver hydroxylase, the percentage of tritium retained in the [methyl-14C]-L-carnitine product was determined as a function of percent reaction. The results of these studies indicated that pro-R hydrogen atom abstraction exceeded 99.9%. Experiments using racemic [methyl-14C,3(RS)-3-3H]-gamma-butyrobetaine as substrate yielded similar results and additionally allowed us to estimate alpha-secondary tritium kinetic isotope effects of 1.10 and 1.31 for the bacterial and calf liver enzymes, respectively. These results are discussed within the context of the radical mechanism for gamma-butyrobetaine hydroxylase previously proposed [Blanchard, J. S., & Englard, S. (1983) Biochemistry 22, 5922], and the required topographical arrangement of enzymic oxidant and substrate is illustrated.

On the mechanism of actomyosin ATPase from fast muscle.

The labeled inorganic phosphate formed by enzymatic hydrolysis of [gamma-18O]ATP in normal water was derivatized to trimethyl phosphate and analyzed for the proportions of [18O3]Pi, [18O2]Pi, [18O1]Pi, and [18O0]Pi. The proportions observed were correlated with the kinetics of intermediate exchange by using a kinetic relationship in which it is assumed that binding of ATP and subsequent release of products are irreversible. Actomyosin and acto-heavy meromyosin catalyze intermediate exchange at a mean rate that is more than 1 order of magnitude slower than that predicted by rapid kinetic studies or implied by the essentially complete intermediate exchange observed with myosin alone. The reason for the slow apparent exchange is that there are two ATPase activities with different exchange properties. The effect of varying heavy meromyosin concentrations at a constant actin concentration shows that the two activities are interrelated and suggests further that one is due to ATP hydrolysis by the undissociated actomyosin crossbridge.

Mechanism of adenosine 5'-triphosphate cleavage by myosin: studies with oxygen-18-labeled adenosine 5'-triphosphate.

During the hydrolysis of MgATP catalyzed by myosin, ATP bound to the protein undergoes a reaction such that the beta-nonbridge oxygen atoms exchange position with the beta gamma-bridge oxygen atom. The extent of this exchange was variable but averaged 45% for ATP that had been bound for 2 s at the myosin subfragment 1 active site at ionic strength 0.08 M, pH 8.0, and 22 degrees C. This result proves that ATP cleavage in the myosin active site is readily reversible. The result also suggests that the beta-phosphate of ADP that must be formed in this cleavage step is highly constrained in the protein.

The stereochemical course of acetate activation by yeast acetyl-CoA synthetase.

The purified alpha-thiophosphate diastereoisomers of adenosine 5'-(1-thio)-triphosphate were used to study the stereochemical course of the reaction catalyzed by yeast acetyl-CoA synthetase. Asymmetrically labeled adenosine 5'-thiophosphate was formed from the "B" diastereoisomer of adenosine 5'-(1-thio)-triphosphate and [18O]acetate. The label was found to be in the opposite orientation from the leaving pyrophosphate group showing that the acetate activation step occurred with inversion of configuration at the alpha-phosphorus.

Specificity of 2-keto-3-deoxygluconate-6-P aldolase for open chain form of 2-keto-3-deoxygluconate-6-P.

2-Keto-3-deoxygluconate-6-P exists as an euqilibrium of three forms at 25 degrees measurable by 13C NMR: alpha-furanose anomer (41%), beta-furanose anomer (50%), and open chain keto (9%). The three forms are interconverted rapidly (greater than 0.5 s-1) so that the unidirectional rates of furanose ring opening and closing can be quantitated by an NMR line broadening method. The 2-keto-3-deoxygluconate aldolase is specific for only one of these forms, the open chain keto form. The rates for ring opening calculated from the rapid kinetic enzyme system compare closely with those obtained with the NMR method.

Studies on the mechanism of Escherichia coli glucosamine-6-phosphate isomerase.

Escherichia coli glucosamine-6-phosphate isomerase is specific for removal of the 1-pro-R hydrogen of fructose 6-phosphate (fructose-6-P). The conversion of [2-3H]glucosamine-6-P to fructose-6-P plus ammonia is accompanied by 99% exchange of tritium with water and 0.6% transfer to C-1 of fructose-6-P. The enzyme is active toward alpha-glucosamine-6-P and apparently inactive toward the beta anomer. The combination of the above results supports a cisenolamine intermediate for the reaction. The labeling of substrate and product pools in tritiated water shows that the two halves of the reaction are each freely reversible. No single step appears to be rate determining. 2-Amino-2-deoxyglucitol-6-P is an unusually strong competitive inhibitor (K1 = 2 X 10(-7) M, compared with the Km = 4 X 10(-4) M for glucosamine-6-P), suggesting the enzyme has a strong affinity for the open-chain form of glucosamine-6-P.

A stereochemical method for detection of ATP terminal phosphate transfer in enzymatic reactions. Glutamine synthetase.

An isotope scrambling method is described for the detection of transient [Enz:ADP:P-X] formation from [18O]ATP in ATP-coupled enzyme reactions. The method makes use of torsional symmetry of the newly formed (see article) group in ADP. [18 O]ATP labeled in the betagama bridge oxygen was incubated with enzyme and reversible cleavage of the PbetaO -- Pgamma bond was detected by the appearance of 18O in the beta nonbridge oxygens of the ATP pool. Experiments with sheep brain and Escherichia coli glutamine synthetases show that cleavage of ATP of enzyme-bound ADP and P-X requires glutamate. The exchange catalyzed by the E. coli enzyme with glutamate occurs in the absence of ammonia and is partially inhibited by added NH4Cl, as expected if the exchange is in the mechanistic pathway for glutamine synthesis. The results provide kinetic support for a two-step mechanism where phosphoryl transfer from ATP to glutamate precedes reaction with ammonia.

Fructose 1,6-bisphosphate: isomeric composition, kinetics, and substrate specificity for the aldolases.

13C NMR shows fructose 6-phosphate and fructose 1,6-bisphosphate to contain respectively 4.1 and 2.0% keto isomer at room temperature. The lower value for fructose 1,6-bisphosphate can be attributed to the electron-withdrawing effect of the C-1 phosphate. Measurements of the ring-opening rates of the alpha and beta anomers of fructose, 1,6-bisphosphate by an NMR line-broadening technique show them to be about 8 and 35 S-1, respectively, at pH 7.2, and 25degreesC. The value for the predominant beta anomer is threefold greater than the turnover rate of muscle aldolase so that, if the kinetic properties of the keto form were favorable, the reaction could proceed entirely through the keto form in solution. The kinetic properties of a fructose 1,6-bisphosphate(keto) analogue, 5-deoxyfructose, 1,6-bisphosphate, in the muscle aldolase reaction are more favorable (Vmax = 2.6, Km = 0.11 X 10(-6) M) than those of fructose 1,6-bisphosphate total (Vmax = 1, Km = 2.3 X 10(-6)M), giving a value of Vmax/Km that is 56 times greater for the 5-deoxy analogue. At the 2.0% concentration of the keto form this is sufficient to account for the steady-state rate and requires that the beta form, present at 40 times greater concentration, contributes little to the cleavage rate. With yeast aldolase the cleavage rate can be explained by the rapid spontaneous ring opening and reaction of the keto form with the enzyme. In view of the high rate of ring opening and the excellent properties of the keto form, previous rapid kinetic studies favoring action of cyclic forms may require reevaluation.

Kinetic demonstration of the intermediate role of aminoacyl-adenylate-enzyme in the formation of valyl transfer ribonucleic acid.

The question whether aminoacyl-tRNA synthetases act in a stepwise or in a concerted mechanism has been investigated kinetically with the valine enzyme of Escherichia coli, which had been used in previous studies by others who concluded that the physiological mechanism is concerted. An exchange between aminoacyl-tRNA and tRNA, dependent upon AMP, was studied. PP-i inhibits this exchange completely in the presence of Mg2+ and AMP but in the absence of added Mg2+ or with dAMP as the nucleotide the inhibition by PP-i is only partial; this is compatible with a stepwise, not a concerted, reaction. Exchange of isotopically labeled substrates in a system at chemical equilibrium also shows effects of substrate concentrations on rates in agreement with the predictions of a stepwise mechanism.

The role of divalent cations in the reactions of valyl transfer ribonucleic acid synthetase of Escherichia coli. Effects of spermine and ethylenediaminetetraacetate.

We have analyzed the function of spermine in the aminoacylation of tRNA-Val by the valyl-tRNA synthetase of Escherichia coli. Our results indicate that Mg2+ is required for the aminoacylation reaction as well as for the ATP-PP-i exchange catalyzed by this enzyme. The apparent stimulation by spermine is a function of the tRNA used, which appears to contain bound cations even after dialysis against 10 minus 4 M EDTA. Higher concentrations of EDTA totally abolish spermine-stimulated esterification of tRNA-Val.

Deamidation in vivo of an asparagine residue of rabbit muscle aldolase.

Microheterogeneity of rabbit muscle aldolase is caused by deamidation in vivo of an asparagine residue near the C-terminus of each subunit. Isotopic labeling of a peptide containing the asparagine residue at various time intervals before isolation of aldolase permits estimation of the half-time for the deamidation as about 8 days, which is about the time estimated for the half-life of the enzyme in vivo. It is concluded that the aldolase as genetically determined is a tetramer, designated alpha(4), that undergoes random deamidation to form alpha(3)beta, alpha(2)beta(2), and alphabeta(3) species as intermediates in the formation of beta(4), the species in which all of the specific asparagine has been deamidated. Isoelectric focusing data indicate that the subunits do not exchange appreciably in vivo.