Akiko Takai

Kinetic isotope effect on the reaction of D-Amino-acid oxidase

Upon anaerobic mixing of D-alanine and D-aminoacid oxidase (EC 1.4.3.3), by rapid reaction technique, transitory appearance of long-wavelength absorbing intermediate was observed [ 1, 21, which was found to be a single species and was identified spectrophotometrically with the purple complex observed in an equilibrium state [2]. The absorbance at 550 nm increases rapidly and subsequently decreases slowly, indicating the rapid formation of the purple intermediate and its slow conversion to the fully reduced enzyme. The reaction sequence in anaerobic reduction of the enzyme with the substrate is expressed as follows [l, 21:

Release of α-imino acid as primary product in d-amino-acid oxidase reaction

Abstract Due to the formation of oxidation products from noncyclic amino acids, such as d -alanine and d -leucine, on catalysis with d -amino-acid oxidase ( d -amino acid:O2 oxidoreductase (deaminating), EC 1.4.3.3) in slightly alkaline solution the pH of the solution decreased and subsequently increased towards the initial level. In the case of cyclic amino acids such as d -proline, on the other hand, only a decrease in pH was observed. These results canbe interpreted to mean that an imino acid, of which the imine group is unprotonated, is released as the primary oxidation product from the enzyme before its hydrolysis to keto acid and NH4+.

The physiology of the smooth muscle: an interdisciplinary review—part II

In the guinea pig taenia coli, when glycogen is depleted by repeating Ca-induced contracture in excess K solution containing no glucose, the tension cannot be maintained. The decrease in tension is accompanied by reduction of high energy phosphate compounds and oxygen consumption. When substrate is readmitted to the glycogen-depleted preparation in the presence of 2.4 mM Ca and 20 mM K, the first response is hyperpolarization of the membrane and relaxation, and this is followed by depolarization and development of contracture. The latter response is blocked by verapamil, suggesting that energy supply increases the Ca conductance of the plasma membrane. The early response is considered to be due to activation of electrogenic Ca pump, since this is not affected by ouabain as well as removal of Na and K. ATP produced by substrate readmission is probably preferentially utilized for Ca pump activation to reduce the intracellular Ca. The recovery of tension is likely to be brought about by ATP supply not only to the contractile machinery but also to the plasma membrane to remove inactivation of Ca conductance. It is postulated that as the energy source is depleted, energy consumption is automatically limited by suppressing Ca influx, as a self-defence mechanism. Since beta HB is as effective as glucose in the recovery of these processes, and also in the activation of electrogenic Na pump, the metabolic pathway of oxidative phosphorylation alone can support these functions without a contribution of the glycolytic pathway.In the guinea pig taenia coli, when glycogen is depleted by repeating Ca-induced contracture in excess K solution containing no glucose, the tension cannot be maintained. The decrease in tension is accompanied by reduction of high energy phosphate compounds and oxygen consumption. When substrate is readmitted to the glycogendepleted preparation in the presence of 2.4 mM Ca and 20 mM K, the first response is hyperpolarization of the membrane and relaxation, and this is followed by depolarization and development of contracture. The latter response is blocked by verapamil, suggesting that energy supply increases the Ca conductance of the plasma membrane. The early response is considered to be due to activation of electrogenic Ca pump, since this is not affected by ouabain as well as removal of Na and K. ATP produced by substrate readmission is probably preferentially utilized for Ca pump activation to reduce the intracellular. Ca. The recovery of tension is likely to be brought about by ATP supply not only to the contractile machinery but also to the plasma membrane to remove inactivation of Ca conductance. It is postulated that as the energy source is depleted, energy consumption is automatically limited by suppressing Ca influx, as a selfdefence mechanism. Since βHB is as effective as glucose in the recovery of these processes, and also in the activation of electrogenic Na pump, the matabolic pathway of oxidative phosphorylation alone can support these functions without a contribution of the glycolytic pathway.

Mechanism of enzyme action: VII. Kinetic analysis of the reaction of d-amino-acid oxidase with d-arginine

1. 1. d-Amino-acid oxidase [d-amino-acid:O2 oxidoreduct ase (deaminating), EC 1.4.3.3], upon anaerobic mixing with d-arginine, was found to be converted from the oxidized to the fully reduced state, but the transient appearance of a purple intermediate, which is characteristic of the reaction with neutral amino acids, was not observed by stopped-flow spectrophotometry. The rate of reduction of the enzyme with d-arginine was directly proportional to the concentration of the amino acid, and the second-order rate constant was calculated to be 390 M−1·s−1 at pH 8.3 and 20 °C. The rate was reduced to about one-fourth by substitution of the α-H of the substrate for deuterium, indicating the rate-limiting removal of the α-H of the substrate. 2. 2. In the reaction of the fully reduced enzyme with O2, no long-wavelength absorbing intermediate was observed. The second-order rate constant of the reaction was 1.9·104 M−1·s−1 at pH 8.3 and 20 °C. 3. 3. The kinetic analysis of the overall reaction of the enzyme-catalyzed oxidation of d-arginine, examined at various concentrations of the substrate and O2, indicated the involvement of a first-order step in the reaction mechanism in addition to the reductive and oxidative steps.

Interaction of chinoform with electron transfer system of rat liver microsomes

Nachweis, dass Chinoform, 5-chloro 7-iodo 8-quinolinol, mit Mikrosomen der Rattenleber weder hydroxyliert noch dehalogeniert werden kann. Inkubation mit Chinoform vermindert die Komponenten des elektronischen Transfersystems, was auch mit in vivo-Versuchen festgestellt wurde.

Mechanism of enzyme action. VI. Kinetic isotope effect on d-amino acid oxidase reaction

Abstract 1. 1. The rate of formation of the purple itermediate of d -amino acid oxidase [ d -amino acid:O2 oxidoreductase (deaminating), EC 1.4.3.3] in the anaerobic reaction of this enzyme with its substrate was reduced by substitution of the α-hydrogen of the substrate for deuterium. This indicates that removal of the α-hydrogen of the substrate occurs prior to or in concert with the formation of the purple intermediate. The extent of the deuterium kinetic isotope effect differed for different substrates. 2. 2. The α-deuteration of the substrate affected neither the rate of conversion of the purple intermediate to the fully reduced enzyme nor the rate of oxidation of the purple intermediate with O2. 3. 3. The kinetic isotope effect on the catalytic oxidation of the substrate was less than that on the formation of the purple intermediate. This indicates that the purple intermediate formation partially determines the overall rate of the enzymic reaction, but the effect on the overall rate is thought to result merely from the effect on the process of the purple intermediate formation. The kinetic isotope effect on the catalytic oxidation was increased by decreasing the enzyme concentration. This may be interpreted to mean that the process of the purple intermediate formation is more rate controlling in the monomeric enzyme than in the dimeric enzyme.

Conversion of the red semiquinone of d-amino acid oxidase to the blue semiquinone by complex formation

Abstract 1. 1. The red semiquinone of d -amino acid oxidase ( d -amino acid:O 2 oxidoreductase (deaminating), EC 1.4.3.3) prepared by photoreduction was changed into the blue semiquinone upon anaerobic addition of excess benzoate. 2. 2. Double reciprocal plots between the concentrations of the blue semiquinone formed and those of benzoate gave a straight line, indicating a complex formation between the semiquinoid enzyme and benzoate. The dissociation constant of the complex was 2.10 −2 M. 3. 3. Both d - and l -alanine combine with the red semiquinone of the enzyme in competition with benzoate. They both form complexes of the red species. 4. 4. The relation between the dissociation constant of the complex (blue species) formed with m - or p -substituted benzoate derivative and the empirical value, Hammetts σ (L. P. Hammett, Physical Organic Chemistry , McGraw-Hill, New York, 1940, p. 184) gave a straight line in the range of σ = −0.4-0, indicating the participation of a COO − group in the formation of the blue species. 5. 5. Anaerobic addition of cinnamate or crotonate to the red semiquinone of the enzyme also gave the blue species.