SH Enzyme Research Articles

Presence of ADP-Glucose Pyrophosphorylase in Shrunken-2 and Brittle-2 Mutants of Maize Endosperm

ADP-Glucose pyrophosphorylase activity has been detected in relatively low amounts in the embryos and endosperms of sh(2) and bt(2) mutant maize seeds. The total enzyme activities in sh(2) and bt(2) were about 12% and 17% respectively, of that found in starchy maize seeds (Dekalb 805). The ADP-glucose pyrophosphorylases from the starchy and mutant maize seeds were activated by 3-phosphoglycerate. However, the extent of the activation of the sh(2) enzyme was not as great as that observed with the bt(2) and Dekalb 805 enzymes. The low levels of ADP-glucose pyrophosphorylase activity in the maize mutants correlate well with the low levels of starch found in the endosperm of these mutants.

Stereoselective reactivity of the SH groups of yeast glyceraldehydephosphate dehydrogenase in the allosteric T and R states

Yeast glyceraldehyde-3-phosphate dehydrogenase as a typical SH enzyme is inactivated by the antipodes of a-iodopropionic acid and its amide at different rates. The apoenzyme reacts faster with the D(+) antipode of the free a-iodopropionic acid ( k D/ k L = 6.8) and the L(−) antipode of the amide ( k L/ k D = 3). On addition of NAD + the stereoselectivity of the SH group towards a-iodopropionic acid is inverted, that towards the amide is enlarged, the rate relationships depending on the NAD + concentration. The results were interpreted by the assumption, that the allosteric T state of the enzyme reacts most rapidly with the D(+) antipodes, whereas the R state favours the L(−) antipodes of the alkylation reagents. The dependence of the reaction rates on the NAD + concentration could be fitted to the allosteric function of state R .

Studies on N5, N10-methenyltetrahydrofolate cyclohydrolase

The enzyme 5,10-methenyltetrahydrofolate cyclohydrolase, which catalyzes the hydrolysis of the above compound to 10-formyltetrahydrofolate, has been purified about 100-fold by ammonium sulfate precipitation and DEAE-cellulose column chromatography. The stability of this extremely unstable enzyme is enhanced by mercaptoethanol and glycerol. Evidence that this is an SH enzyme was derived from the inhibitory effects of standard sulfhydryl agents and the partial activation by reducing compounds. Consonant with this is the inhibition observed with the metal ions Cu ++, Cd ++, Hg ++, Fe ++, and Fe ++. The optimum pH (8.0), temperature stability, and Michaelis constants for the reaction in the forward and reverse direction have been determined and utilized to estimate the equilibrium constant of the reaction. At concentrations higher than 35 μM 5,10-methenyltetrahydrofolate and 10-formyltetrahydrofolate were found to inhibit the enzyme.

THE METABOLISM OF L-RHAMNOSE IN ESCHERICHIA COLI. II. L-RHAMNULOSE KINASE.

Abstract Extracts of Escherichia coli , prepared from cells grown on l -rhamnose, contained a l -rhamnulose kinase (ATP: l -rhamnulose i -phosphotransferase, EC 2.7.1.5) which has been purified about 13-fold. The enzyme appears to be an SH enzyme, which catalyzes the phosphorylation of l -rhamnulose in the presence of ATP and some divalent metal ions but seems not to reactive with other sugars. Analytical studies on the phosphorulated sugar suggest that the reaction product is l -rhamnulose i -phosphate. Evidence is present which indicates that the first step in the fermentation of l -rhamnose is the conversion of the methylpentose to l -rhamnulose followed by a phosphorylation of the ketose.

THE METABOLISM OF L-RHAMNOSE IN ESCHERICHIA COLI. I. L-RHAMNOSE ISOMERASE.

Abstract l -Rhamnose isomearse ( l -rhamnose ketol-isomerase) has been purified about 50-fold from extracts of Escherichia coli . THe enzyme appears to be an SH enzyme, and catalyzes the reversible reaction l -rhamnose ⇆ l -rhamnulose. The enzyme was activated by the addition of manganous ions. At equilibrium about 60% of the total methylpentose was present as l -rhamnulose. The enzyme was specific for l -rhamnose and l -rhamnulose; no other sugars were found to react.

Studies on Aspartic ^|^beta;-Semialdehyde Dehydrogenase in Pea Seedlings

Highly purified aspartic β-semialdehyde dehydrogenase was prepared from pea seedlings. The optimum pH and Km values for the substrates were determined, and the substrate and coenzyme specificities of this enzyme were examined.Effects of various compounds on aspartic β-semialdehyde dehydrogenase prepared from pea seedlings were examined. From the behaviours toward thioglycolate and PCMB, this enzyme was concluded to be a -SH enzyme. The inhibitory effects of aldehydes and amino acids were also studied.

Studies on Microbial Biosynthesis of Pantothenic Acid

Pantothenate synthesis from pantoate and β-alanine by Escherichia coli extracts was studied as a model synthesis of the peptide bond. It was found as the evidence for occurence of pantoyl-carboxyl activation that a significant quantity of pantoic acid hydroxamate was formed in the reaction mixture containing pantoate and ATP. Formation of pantothenate and development of hydroxamate color from activated pantoate were increased under the anaerobical conditions. Inhibition by -SH reagents and reversal of inhibition by thiol compounds are indicative of the evidence for involvement of -SH enzyme in pantothenate synthesis and pantoate activation. In view of these results, pantoate activation is formulated as shown in the following two-step reaction: HS−E+AMP~PP⇄S·E+AMP PP~S·E+C5H11O2·COO-⇄C5H11O2·CO~S·E+PP.

Studies on Microbial Biosynthesis of Pantothenic Acid

Pantothenate synthesis from pantoate and β-alanine by Escherichia coli extracts was studied as a model synthesis of the peptide bond. It was found as the evidence for occurence of pantoyl-carboxyl activation that a significant quantity of pantoic acid hydroxamate was formed in the reaction mixture containing pantoate and ATP. Formation of pantothenate and development of hydroxamate color from activated pantoate were increased under the anaerobical conditions. Inhibition by -SH reagents and reversal of inhibition by thiol compounds are indicative of the evidence for involvement of -SH enzyme in pantothenate synthesis and pantoate activation. In view of these results, pantoate activation is formulated as shown in the following two-step reaction: HS−E+AMP~PP⇄S·E+AMPPP~S·E+C5H11O2·COO-⇄C5H11O2·CO~S·E+PP.

Effect of acetoacetate on the succinic acid oxidase system.

THE effect of alloxan, a well-known diabetogenic substance, on blood glutathione1 and enzymes2 indicates that it is possible that its diabetogenic action may be through its effect on enzyme systems. Lazarow3 has discussed this possibility at length, and has also suggested that acetoacetic acid or a related compound may have a direct toxic action on the enzymes in the beta-cells of the pancreas and may thereby have an alloxan-like action. The recent findings reported from this laboratory4 that blood glutathione is lowered by acetoacetate injection may serve as an additional indication of acetoacetate acting adversely on sulphydryl enzymes. In a preliminary observation on a simple —SH enzyme papain5, it was noticed that acetoacetate could completely inactivate the enzyme at a concentration of 16 mM.

Effect of X-radiation upon succinoxidase of rat kidney.

Summary1. The succinoxidase system, as well as its component enzymes, the -SH enzyme succinic dehydrogenase, and cytochrome oxidase, were determined in rat kidney homogenates within two hours after rats were X-irradiated with 400 r or 800 r. No inhibition of any of the enzymes was found. 2. The metabolism of kidney slices from rats given the above amounts of X-radiation was studied manometrieally with sodium succinate and lithium pyruvate substrates. No statistically significant differences were obtained between the oxidative activities of the kidney slices from the control and irradiated animals. 3. The lack of correlation between certain of the in vivo and in vitro effects of irradiation upon enzyme systems is discussed.