Selective arylation of cysteine-237 of rabbit muscle aldolase with 4-chloro-7-nitrobenzofurazan.

At pH 7.0 and 25 degrees C, NBF-Cl (4-chloro-7-nitrobenzofurazan) reacts rapidly with rabbit muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) to yield a product with an absorption maximum at 402 nm, which is shifted to 422 nm upon acid denaturation. The reaction involves arylation of a single cysteine residue per subunit of tetrameric aldolase, as shown by the molar absorptivity of NBF-aldolase and by titration of sulfhydryl groups of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid), DTNB. The site of arylation appears to be Cys-237, which is the cysteine residue that reacts most rapidly with DTNB. The site of arylation appears to be Cys-237, which is the cysteine residue that reacts most rapidly with DTNB, and which is not essential for aldolase activity. Arylation of the enzyme is 13-20-times more rapid than that of model compounds. The relatively high rate of arylation is not due to medium effects, to an anomalously low pKa of Cys-237, or to the presence of a binding site for NBF-Cl, and is tentatively assigned to acid-base catalysis by other functional groups in the vicinity of the reactive sulfhydryl group. The NBF-Cl reaction provides the most efficient means of titrating Cys-237 residues in rabbit muscle aldolase.

Structural and mechanistic similarities of 6-phosphogluconate and 3-hydroxyisobutyrate dehydrogenases reveal a new enzyme family, the 3-hydroxyacid dehydrogenases.

Rat 3-hydroxyisobutyrate dehydrogenase exhibits significant amino acid sequence homology with 6-phosphogluconate dehydrogenase, D-phenylserine dehydrogenase from Pseudomonas syringae, and a number of hypothetical proteins encoded by genes of microbial origin. Key residues previously proposed to have roles in substrate binding and catalysis in sheep 6-phosphogluconate dehydrogenase are highly conserved in this entire family of enzymes. Site-directed mutagenesis, chemical modification, and substrate specificity studies were used to compare possible mechanistic similarities of 3-hydroxyisobutyrate dehydrogenase with 6-phosphogluconate dehydrogenase. The data suggest that 3-hydroxyisobutyrate and 6-phosphogluconate dehydrogenases may comprise, in part, a previously unrecognized family of 3-hydroxyacid dehydrogenases.

Helix stop signals in proteins and peptides: the capping box.

The alpha-helix [Pauling, L., Corey, R. B., & Branson, H. R. (1951) Proc. Natl. Acad. Sci. U.S.A. 37, 205-211] is a common motif in both proteins and peptides. Despite intense investigation, predictive understanding of helices is still lacking. A recent hypothesis [Presta, L. G., & Rose, G. D. (1988) Science 240, 1632-1641] proposed that the structural specificity of helices resides, in part, in those residues that flank helix termini. If so, then signals that arrest helix propagation--i.e., helix stop signals--should be found among these flanking residues. Evidence is presented for the existence of one such signal, a reciprocal backbone-side-chain hydrogen-bonding interaction, dubbed the capping box. In proteins, the capping box is found uniquely at helix N-termini. In peptides, the capping box can function as a helix stop signal, as shown in the work of Kallenbach and co-workers.

Slow reversible inhibition of rabbit muscle aldolase by D-erythrulose 1-phosphate.

Rabbit muscle aldolase was found to be inactivated in a slow, reversible manner by D-erythrulose 1-phosphate. This compound combined rapidly and reversibly with the enzyme to form an initial complex, which then only slowly (ki = 0.28 min-1) converted to a kinetically more stable form. This stable enzyme-ligand form was inactive toward the normal substrate of aldolase, fructose 1,6-bisphosphate. The inactive enzyme-ligand complex, however, could be decomposed (kr = 0.0041 min-1) to yield active enzyme once again by incubation in a solution devoid of D-erythrulose 1-phosphate.

Ion-affinity electrophoresis. Preparation and properties of phosphorylated polyacrylamide gels.

Strong, pliable polyacrylamide gels containing covalently bound phosphate groups have been formed by radical-initiated copolymerization of acrylamide and a phosphorylated, N-substituted derivative of acrylamide. Under conditions of disc electrophoresis, the gels provide enhanced resolution in the separation of hemoglobin A from S, and separate two forms of yeast glucose 6-phosphate dehydrogenase and rabbit muscle glyceraldehyde 3-phosphate dehydrogenase. Preliminary evidence suggests that the improved resolution is due to ionic adsorption of the proteins to immobilized phosphate groups, implying that the gels may function as supports for electrophoretically powered separations involving ion-exchange or affinity chromatography.

Effects of ethanol on cocaine metabolism: formation of cocaethylene and norcocaethylene.

The coabuse of cocaine and ethanol occurs with high frequency and increases the risk of cocaine-related morbidity and mortality. The mechanisms mediating the toxic interactions of cocaine and ethanol are not clearly defined. This study examined the effects of acute ethanol administration on the metabolism of cocaine in the male Wistar rat. Intraperitoneal administration of 2 g/kg ethanol 30 min prior to administration of 25 mg/kg cocaine resulted in the formation of two ethylated derivatives of cocaine, benzoylecgonine ethyl ester (cocaethylene) and benzoylnorecgonine ethyl ester (norcocaethylene) in liver, brain, and serum. Fifteen minutes after cocaine administration, the tissue levels of cocaethylene were 22, 10, and 9% of the cocaine recovered from liver, serum, and brain, respectively. Ethanol pretreatment increased cocaine concentrations in liver and benzoylnorecgonine concentrations in liver and serum. The increased morbidity and hepatotoxicity seen with acute combined administration of cocaine and ethanol may be due to the formation of the toxic ethylated and N-demethylated metabolites of cocaine. Ethanol pretreatment decreased benzoylecgonine concentrations in serum and liver. The most important consequence of ethanol-induced inhibition of the normally rapid hydrolysis of cocaine to benzoylecgonine may be a decrease in benzoylecgonine-mediated vasoconstriction.

Primary structure and tissue-specific expression of human beta-hydroxyisobutyryl-coenzyme A hydrolase.

beta-Hydroxyisobutyryl-CoA (HIBYL-CoA) hydrolase is responsible for the specific hydrolysis of HIBYL-CoA, a saline catabolite, as well as the hydrolysis of beta-hydroxypropionyl-CoA, an intermediate in a minor pathway of propionate metabolism. We have obtained the amino acid sequences of several tryptic peptides derived from purified rat liver HIBYL-CoA hydrolase, and the NH2-terminal peptize sequence was matched to the translated sequence of a human expressed sequence tag present in the data base of the IMAGE Consortium (Lawrence Livermore National Laboratory, Livermore, CA). The complete nucleotide sequence and the deduced amino acid sequence showed no similarity to the sequences of well known thioesterases but showed significant homology to the enoyl-CoA hydratase/isomerase enzyme family. The cDNA fragment corresponding to the mature (processed) protein was expressed in Escherichia coli. The purified recombinant enzyme displayed substrate specificity very similar to that of the rat enzyme and was specifically bound by polyclonal antibodies raised against purified rat liver HIBYL-CoA hydrolase. Northern and Western blot analyses with various human tissues indicated predominant expression in liver, heart, and kidney, with discrepancies occurring in the amounts of HIBYL-CoA hydrolase mRNA compared to stably expressed protein in several tissues.