ROLE AND REACTIVITY OF SULFHYDRYL GROUPS IN FIREFLY LUCIFERASE

被引:36
作者
LEE, R
MCELROY, WD
机构
[1] Department of Biology, McCollum-Pratt Institute, Johns Hopkins University, Baltimore, Maryland
关键词
D O I
10.1021/bi00829a018
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
The chloromethyl ketone derivative of N tosyl-L-phenylalanine is an effective inhibitor of firefly luciferase. The inhibition of enzymatic activity is accompanied by a loss of approximately two sulfhydryl groups. The chloromethyl ketone derivative of N-tosyl-L-lysine is without effect under similar conditions. Kinetically, N-tosyl-L-phenylalanine chloromethyl ketone inhibition is competitive with respect to one of the substrates, luciferin, and noncompetitive with respect to the other, adenosine 5′-triphosphate. The hydrophobic character of N-tosyl-L-phenylalanine chloromethyl ketone appears to be the major factor for its binding to the active site of luciferase. The fact that N-tosyl-L-phenylalanine is a competitive inhibitor also with a Kiof the same order of magnitude as N-tosyl-Lphenylalanine chloromethyl ketone supports this conclusion. It appears, therefore, that N-tosyl-L-phenylala nine chloromethyl ketone inhibition of luciferase involves two phases: first, a reversible binding of N-tosyl-L-phenylalanine chloromethyl ketone molecules at the luciferin binding sites (demonstrable kinetically as competitive inhibition), and second, the reaction of the chloromethyl ketone group of N-tosyl-L-phenylalanine chloromethyl ketone with the SH groups at or near these sites. Inactivation by N-tosyl-L-phenylalanine chloromethyl ketone is pH dependent; inactivation- pH curve corresponds very closely to the luciferase activity-pH curve. It is suggested that two sulfhydryl groups are located at or near the binding site of luciferin, and that they are required in the luciferase-catalyzed production of yellow-green light. © 1969, American Chemical Society. All rights reserved.
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[21]   STRUCTURE AND SYNTHESIS OF FIREFLY LUCIFERIN [J].
WHITE, EH ;
FIELD, GF ;
MCELROY, WD ;
MCCAPRA, F .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1961, 83 (10) :2402-&