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1. I. The reduction of riboflavin by dithionite was followed, using a rapid mixing apparatus, over a range of concentration, acidity and temperature. 2. 2. The time-course of reduction of riboflavin in the presence of excess dithionite was found not to be of exponential form, but of approximately linear form. Evidently the reaction is complex. 3. 3. In sufficiently dilute riboflavin solutions, the initial reaction velocity was directly proportional to riboflavin concentration. Under these conditions, the Q10 was approximately 2.0. 4. 4. In sufficiently concentrated riboflavin solutions, the initial reaction velocity became nearly independent of riboflavin concentration. 5. 5. An intermediate was found by its absorption in the infra-red. The maximum amount of intermediate formed was proportional to the square of the riboflavin concentration. The intermediate was only detected in sufficiently concentrated riboflavin solutions. Smaller amounts of intermediate were formed at higher temperatures. 6. 6. The mechanism of the reaction is discussed.

The inhibition of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) by L-hydrazinosuccinate has been studied. The velocity of the enzyme reaction decreased with time when the reaction was initiated by the addition of enzyme to a mixture of the assay components and L-hydrazinosuccinate, while it increased slowly from a low level when a preincubated mixture of the enzyme and the inhibitor was added to the reaction mixture to initiate the reaction. Nearly 50% decrease in the initial reaction velocity was produced by a prolonged preincubation of the enzyme with the inhibitor, both at low concentrations of about 2 nM. These findings indicate that the inhibition is of the slow- and tight-binding type. The time-course of the reaction of the enzyme and the inhibitor, examined by the change in activity, was not in accord with single-step mechanisms, but rather appeared to follow biphasic kinetics. The inhibition could be fully reversed only in the presence of L-cysteine sulfinate or large excess of L-aspartate to convert the regenerated enzyme to its pyridoxamine form. The time-course of the reversal followed pseudo-first-order kinetics. Quantitative analysis of the experimental data has shown that the results are consistent with a mechanism of enzyme-inhibitor interaction which involves a reaction of two consecutive, reversible steps. The overall inhibition constant for L-hydrazinosuccinate was calculated to be approx. 0.2 nM.

Bilirubin was found to be a potent inhibitor of NAD+-linked isocitrate dehydrogenase (threo-ds-isocitrate:NAD+ oxidoreductase (decarboxylating), EC 1.1.1.41). It inhibited the enzyme by decreasing the affinity of enzyme for isocitrate, while the maximum reaction velocity was not affected. Bilirubin (less than 10−6 M) showed a detectable inhibition. On the other hand, neither the mitochondrial nor the cytoplasmic NADP+ enzymes (threo-ds-isocitrate:NADP+ oxidoreductase ((decarboxylating), EC 1.1.1.42) were affected by bilirubin.

1. 1. Some aspects of the kinetics of the CPK catalysed reaction PC + ADP ⇌ ATP + creatine and the effects of certain arsenical inhibitors have been studied. 2. 2. CPK is activated by Ca +2 , Mg +2 and Mn +2 , and the reaction velocity is greatest in the presence of the latter ion. 3. 3. The effects of lewisite and diphenylchloroarsine suggest that CPK is a “monothiol” enzyme.

Squid rhodopsin made alkaline with borate-NaOH buffer is bleached by irradiation with yellow light. After irradiation, rhodopsin is regenerated from a substance with an absorption maximum of 380 nm (P 380 ). This regeneration of rhodopsin is scarcely observed in a solution made alkaline with sodium carbonate instead of borate buffer. The reaction velocity of the conversion of P 380 to rhodopsin is dependent not only on temperature and pH but also on the concentrations of borate buffer and digitonin. P 380 is produced from acid metarhodopsin during irradiation with yellow light and its chromophore must be 11- cis -retinal. This substance with λ max of 380 nm is an intermediate in the photoregeneration process of squid rhodopsin.

Summary The monovalent cation requirements of formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase (ADP), EC 6.3.4.3) and the mechanisms by which di-and monovalent cations activate the enzyme were examined. The enzyme shows an absolute requirement for a monovalent cation. The relative effectiveness of the cations tested is NH 4 + >K + = Rb + >Cs + >Na + >Li + . It was found that the K m values of formate and tetrahydrofolate increased as the concentration of NH 4 + was lowered from 50 to 0.8 mM. The same change in NH 4 + had no effect on the maximum velocity of the reaction at infinite concentrations of either formate or tetrahydrofolate. On the other hand the maximum velocity at infinite ATP was influenced by NH 4 + concentrations. The results indicate that the enzyme binds with the monovalent cation before tetrahydrofolate and formate. The binding of ATP is, however, independent of NH 4 + . Spermine can replace NH 4 + to a certain degree but does not stimulate the reaction velocity in the presence of a saturating amount of NH 4 Cl. In addition, the results of kinetic data support the view that Mg 2+ activation of the enzyme results from complex formation between ATP and Mg 2+ to form the substrate, MgATP * .