The catalytic mechanism of transketolase. Thiamin pyrophosphate-derived transition states for transketolase and pyruvate dehydrogenase are not identical.

Thiamin thiazolone pyrophosphate (TTPP) has been reported to be an effective transition state analogue for the thiamin pyrophosphate-dependent partial reaction of pyruvate dehydrogenase (Gutowski, J. A., and Lienhard, G. E. (1976) J. Biol. Chem. 251, 2863-2866). The kinetics of the interaction of TTPP with transketolase are reported here. TTPP is a competitive inhibitor, with respect to thiamin pyrophosphate, of bakers' yeast transketolase but it is neither a tight binding inhibitor nor a slow binding inhibitor. TTPP decreases the kinetically observed negative cooperativity seen for thiamin pyrophosphate and also decreases the rate constant for the hysteretic activation of the enzyme by thiamin pyrophosphate. We conclude that thiamin thiazolone pyrophosphate is not an effective transition state analogue for the reaction catalyzed by bakers' yeast transketolase. This difference between transketolase and pyruvate dehydrogenase may be related to differences in the polarity of the active sites of the enzymes. It is conceivable that the active sites of the pyruvate decarboxylase subunit of pyruvate dehydrogenase is hydrophobic, by analogy with the known hydrophobicity of the active site of brewers' yeast pyruvate decarboxylase. This hydrophobicity would stabilize a transition state with no charge on the thiazole portion of the coenzyme, similar to the "uncharged" thiazole portion of TTPP. In contrast, the active site of bakers' yeast transketolase, which is known to contain charged amino acid side chains, should be less favorable for such an uncharged transition state. A charge-separated canonical form related to TTPP could be preferentially stabilized in the active site of transketolase.

Two glutamine synthetases from Bacillus caldolyticus, an extreme thermophile. Isolation, physicochemical and kinetic properties.

Two distinctly different glutamine synthetase enzymes (EI and EII) have been isolated from the extreme thermophile Bacillus caldolyticus, grown on chemically defined medium at 70 degrees C. Purification to homogeneity mainly involves affinity chromatography and heat treatment with substrate protection. Biosynthesis of total enzyme activity can be repressed by at least 8-fold by high ammonia, with synthesis of EI being repressed more strongly than EII. A variety of chemical and biochemical tests failed to provide evidence for regulation of EI or EII by covalent modification, e.g. proteolysis, phosphorylation, or adenylylation. Neither of the thermophiic enzymes will cross-react with antibodies for the Escherichia coli or Bacillus subtilis glutamine synthetases. Both enzymes are composed of 12 subunits, each approximately 51,000 daltons. However, EI and EII differ significantly in their amino acid composition, isoelectric points (5.2 and 5.5, respectively), rates of migration on polyacrylamide electrophoresis gels at pH 6.8, and kinetic properties, EI is more active with Mg(II) than with Mn(II), but EII is more active with Mn(II) than Mg(II). Cd(II) activates EII more than EI, and only EI shows activity with Co(II). For both enzymes, the Mn(II)-stimulated activity is optimal at pH 6.0 to 6.5, with Mn(II)/ATP = 1.0, but the pH optimum with Mg(II) is near pH 7.5, however, with a ratio of Mg(II)/ATP > 2. Substrate Km values at 70 degrees C differ for EI versus EII but are quite comparable to those seen for mesophilic glutamine synthetases. Studies with structural analogs of substrates indicate that active site specificity is maintained at extreme temperatures: substitution of alpha-OH for alpha-HN2 is allowed, but unfavorable changes occur upon substitution of methyl groups for the alpha-H or onto the alpha-NH2 of L-Glu, and for D-Glu or L-Asp. EII is almost absdolutely specific for ATP, but EI can also use ITP, GTP, and UTP as substrates to some extent. The divalent metal ion that is present can affect both specificity for analogs and substrate Km values. Kinetic binding plots (v versus [S]) are biphasic for NH3 and L-Glu with the more active forms of each enzyme, EI-Mg and EII-Mn, respectively; but no positive cooperativity is observed. ATP binding is strictly hyperbolic, in contrast to the positive cooperativity previously observed with other Bacillus sp. enzymes. For purified EI and EII, Arrhenius plots are nonlinear with Mn(II) or Mg(II), exhibiting slope changes in the range of 55-65 degrees C; however, for EI-EII mixtures in crude cell extracts these plots are nearly linear.

Glucose-6-phosphate dehydrogenase from lactating rat mammary gland and R3230AC adenocarcinoma.

A number of properties of glucose-6-phosphate dehydrogenase from lactating rat mammary gland and R3230AC rat mammary adenocarcinoma are compared. The main electrophoretic forms of the enzyme from these sources are indistinguishable with respect to charge and molecular weight whereas the minor forms show differences in these properties. The subunit molecular weight and steroid inhibition of the enzymes from the lactating gland and tumor are not significantly different. These results are contrasted with similar studies in mice.