EVIDENCE FOR SULFHYDRYL-GROUPS AT ACTIVE-SITE OF CATECHOL-O-METHYLTRANSFERASE

被引:14
作者
BORCHARDT, RT [1 ]
THAKKER, DR [1 ]
机构
[1] UNIV KANSAS, DEPT BIOCHEM, MCCOLLUM LABS, LAWRENCE, KS 66045 USA
关键词
D O I
10.1016/0005-2744(76)90113-3
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
Earlier studies using affinity labeling reagents have suggested the existence of 2 nucleophilic groups at the active site of [rat liver] catechol-O-methyltransferase (S-adenosyl-L-methionine:catechol O-methyltransferase, EC 2.1.1.6). Both nucleophilic residues are critical for catalytic activity. In an effort to elucidate the nature of these residues and to further characterize the relationship between the chemical structure and the catalytic function of this enzyme, inactivation studies using N-ethylmaleimide were undertaken. Inactivation of the enzyme by N-ethylmaleimide under pseudo 1st-order conditions exhibited a non-linear relationship between the log of the fraction of enzyme activity remaining and preincubation time. Kinetic analysis of this inactivation process suggested the modification by N-ethylmaleimide of 2 residues at the active site of the enzyme, both crucial for catalytic activity. Detailed analysis of the inactivation process including substrate protection studies, pH profiles of inactivation, and incorporation studies using N-ethyl[2,3-14C2]maleimide provided additional evidence to support this conclusion.
引用
收藏
页码:598 / 609
页数:12
相关论文
共 15 条
[11]   SULFUR ANALOGS OF DOPAMINE AND NOREPINEPHRINE - INHIBITION OF CATECHOL-O-METHYLTRANSFERASE [J].
LUTZ, WB ;
WITKOP, B ;
DALY, JW ;
CREVELIN.CR ;
GOLDBERG, LI .
JOURNAL OF MEDICINAL CHEMISTRY, 1972, 15 (08) :795-&
[12]  
NOTARI RE, 1971, BIOPHARMACEUTICS PHA, P129
[13]  
RAY WJ, 1961, J BIOL CHEM, V236, P1973
[15]  
WEBER K, 1969, J BIOL CHEM, V244, P4406