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D-3-Phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis has been isolated to homogeneity and displays an unusual relationship to the Escherichia coli and mammalian enzymes. In almost all aspects investigated, the M. tuberculosis enzyme shares the characteristics of the mammalian PGDHs. These include an extended C-terminal motif, substrate inhibition kinetics, dependence of activity levels and stability on ionic strength, and the inability to utilize ␣-ketoglutarate as a substrate. The unique property that the M. tuberculosis enzyme shares with E. coli PGDH that it is very sensitive to inhibition by L-serine, with an I 0.5 ‫؍‬ 30 M. The mammalian enzymes are not inhibited by L-serine. In addition, the cooperativity of serine inhibition appears to be modulated by chloride ion, becoming positively cooperative in its presence. This is modulated by the gain of cooperativity in serine binding for the first two effector sites. The basis for the chloride modulation of cooperativity is not known, but the sensitivity to serine inhibition can be explained in terms of certain amino acid residues in critical areas of the structures. The differential sensitivity to serine inhibition by M. tuberculosis and human PGDH may open up interesting possibilities in the treatment of multidrug-resistant tuberculosis.D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) 1 has been investigated in a variety of organisms (1-4) and is a member of a family of proteins classified as 2-hydroxyacid dehydrogenases that are generally specific for substrates with a D-configuration (5). PGDH catalyzes the first committed step in the phosphorylated pathway of L-serine biosynthesis by converting D-3-phosphoglycerate to hydroxypyruvic acid phosphate utilizing NAD ϩ as a cofactor (1, 6). Subsequently, hydroxypyruvic acid phosphate is converted to phosphoserine by phosphoserine transaminase and then to L-serine by phosphoserine phosphatase (6). The reaction catalyzed by PGDH occurs spontaneously in vitro in the direction of conversion of hydroxypyruvic acid phosphate to phosphoglycerate.In some organisms, such as Escherichia coli (7), PGDH is strongly inhibited by L-serine (I 0.5 ϭ ϳ2-4 M), the end product of the pathway. E. coli PGDH is the most studied and is classified as a V-type enzyme (8) because L-serine regulates catalysis by altering the velocity of the reaction rather than the affinity of the substrate. In Bacillus subtilis, inhibition of PGDH by L-serine is less sensitive (I 0.5 ϳ 0.6 mM) but appears to lose its sensitivity to L-serine under oxidizing conditions (9). PGDH from Corynebacterium glutamicum has also been reported to be inhibited by L-serine only at very high concentrations (I 0.5 ϳ 10 mM) but has not been studied in homogeneous form (10). In addition, L-serine inhibition of both the B. subtilis and C. glutamicum PGDH require extensive preincubation of the enzyme with the inhibitor before appreciable inhibition can be measured. In the pea (Pisum sativum), the sensitivity to L-serine has been reported to be cold ...

Sulfite-resistant and sulfite-sensitive mutants of Saccharomyces cerevisiae were isolated and characterized. Genetic analysis indicated that one and four genes were responsible for the resistant and sensitive responses, respectively, and suggested that defects in methionine and cysteine metabolism were not involved. Some resistant alleles, all of which were dominant, conferred greater resistance than others. Mutations conferring sensitivity were recessive and one co-segregated with impaired respiration. Two of the sensitive mutants exhibited cross-sensitivity to other metabolic inhibitors: sulfometuron methyl, cycloheximide, oligomycin, and antimycin A. A 50% glutathione deficiency in one sensitive mutant was not sufficient in itself to account for its sensitivity. Screening of other relevant mutants revealed that relative to wild-type, met8 and a thioredoxin null mutant are sensitive, and met3 and met14 mutants are not. Reduced production of extracellular acetaldehyde, a compound that detoxifies sulfite, was observed in three of the four sensitive mutants. However, acetaldehyde was also underproduced in the resistant mutant. Because sulfite is a reducing agent, cells were tested for coincident sensitivity or resistance to ascorbate, selenite, dithiothreitol, nitrite, thiosulfate, reduced glutathione, and cysteine. No consistent pattern of responses to these agents emerged, suggesting that the response to sulfite is not a simple function of redox potential.

kappa-Bungarotoxin, a kappa-neurotoxin derived from the venom of the banded Krait, Bungarus multicinctus, is a homodimeric protein composed of subunits of 66 amino acid residues containing five disulfide bonds. kappa-Bungarotoxin is a potent, selective, and slowly reversible antagonist of alpha3 beta2 neuronal nicotinic acetylcholine receptors. kappa-Bungarotoxin is structurally related to the alpha-neurotoxins, such as alpha-bungarotoxin derived from the same snake, which are monomeric in solution and which effectively antagonize muscle type receptors (alpha1 beta1 gamma delta) and the homopentameric neuronal type receptors (alpha7, alpha8, and alpha9). Like the kappa-neurotoxins, the long alpha-neurotoxins contain the same five conserved disulfide bonds, while the short alpha-neurotoxins only contain four of the five. Systematic removal of single disulfide bonds in kappa-bungarotoxin by site-specific mutagenesis reveals a differential role for each of the disulfide bonds. Removal of either of the two disulfides connecting elements of the carboxy terminal loop of this toxin (Cys 46-Cys 58 and Cys 59-Cys 64) interferes with the ability of the toxin to fold. In contrast, removal of each of the other three disulfides does not interfere with the general folding of the toxin and yields molecules with biological activity. In fact, when either C3-C21 or C14-C42 are removed individually, no loss in biological activity is seen. However, removing both produces a polypeptide chain which fails to fold properly. Removal of the C27-C31 disulfide only reduces the activity of the toxin 46.6-fold. This disulfide may play a role in specific interaction of the toxin with specific neuronal receptors.

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