1992 Fiscal Year Final Research Report Summary
Study on the Enzymatic Reaction Mechanism catalyzed by Histidine Ammonia-Lyase Using Stable Isotope Methodology
| Project/Area Number |
03807143
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Physical pharmacy
|
|---|
| Research Institution | Tokyo College of Pharmacy |
|---|
Principal Investigator |
FURUTA Takashi Tokyo College of Pharmacy, Pharmacy, Associate Professor, 薬学部, 助教授 (70120152)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
SHIBASAKI Hiromi Tokyo College of Pharmacy, Pharmacy, Assistant, 薬学部, 助手 (20206121)
KASUYA Yasuji Tokyo College of Pharmacy, Pharmacy,Professor, 薬学部, 教授 (90096686)
|
|---|
| Project Period (FY) |
1991 – 1992
|
| Keywords | L-Histidine / Urocanic Acid / Histidine Ammonia-lyase / beta-Elimination / Enzymatic Reaction / Rate-limiting Step / Reversibility / Deuterium Isotope Effect |
|---|
| Research Abstract |
L-Histidine labeled with deuterium at C-5' of the imidazole ring, L-[5'-^2H]histidine, was used as a probe for investigating an enzymatic reaction mechanism in the elimination of ammonia catalyzed by histidine ammonia-lyase (EC 4.3.1.3). The labeled L-histidine was incubated with histidine ammonia-lyase from Pseudomonas fluorescens at pH 7.0 or pH9.0 at 25.0 ﾟC for 24 h. The time course of the reaction was examined to determine the rates of enzyme-catalyzed hydrogen exchange at C-5' of L-histidine and urocanic acid. The finding of the enzyme-catalyzed hydrogen exchange at C-5' of both L-histidine and urocanic acid provided a rational explanation for a stepwise reversible mechanism via a carbanion intermediate in the elimination reaction. The stability of carbanion was also demonstrated to be approximately three times higher at pH 7.0 than at pH 9.0.
The deuterium isotope effects for the histidine ammonia-lyase reaction have also been studied under the various pH conditions (pH 7.0-10.5) by using both L-[3,3-^2H_2]histidine and L-[5'-^2H]histidine. Comparison of the Michaelis constants with labeled (L-[3,3-^2H_2]histidine) and unlabeled substrates revealed that the isotope effects on V_<max> and V/K were 1.00-1.38 and 1.18-1.63, respectively, indicating pH dependent. As the pH was increased,^DV increased from 1.12 at pH 7.0 to 1.38 at pH 8.0 and then decreased above pH 8.0. The isotope effect on V_<max> observed at the optimal pH of 9.0 was 1.28. This indicates that the C_3-H bond cleavage step at pH 8.0 is more rate-limiting and that the rate-limiting step changes as a function of pH. However, no significant isotope effect on the C_5-H bond cleavage step was observed in the reaction of L-[5'-^2H]histidine as substrate.
|
