KINETIC ISOTOPE EFFECTS ON SUBSTRATE ASSOCIATION - REACTIONS OF PHOSPHOENOLPYRUVATE WITH PHOSPHOENOLPYRUVATE CARBOXYLASE AND PYRUVATE-KINASE

被引:21
作者
GAWLITA, E
CALDWELL, WS
OLEARY, MH
PANETH, P
ANDERSON, VE
机构
[1] TECH UNIV LODZ,DEPT CHEM,PL-90924 LODZ,POLAND
[2] UNIV NEBRASKA,DEPT BIOCHEM,LINCOLN,NE 68583
[3] CASE WESTERN RESERVE UNIV,DEPT BIOCHEM,CLEVELAND,OH 44106
关键词
D O I
10.1021/bi00008a023
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Kinetic isotope effects on association have been measured using the remote label methodology developed by O'Leary and Marlier (1979). The isotope effect on V/K-A for the first substrate in an obligatorily ordered mechanism is an isotope effect on its second-order rate constant for association with the enzyme. With phosphoenolpyruvate carboxylase the (18)(V/K-PEP) when the bridging O is labeled decreases from 1.0056 +/- 0.0007 to 0.9943 +/- 0.0002 as the concentration of bicarbonate, the second substrate, increases from 2 to 200 mM. With pyruvate kinase the (18)(V/K-PEP) decreases from 1.0024 +/- 0.0014 to 0.9928 +/- 0.0027 as the concentration of ADP increases from 1.5 to 30 mM. These inverse kinetic isotope effects are best understood as arising from an isotope effect on the rate constant for forming the Michaelis complex of enzyme and substrate. The inverse value suggests that the bridging oxygen is in a vibrationally stiffer environment in the transition state for the association reaction.
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收藏
页码:2577 / 2583
页数:7
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