An analytical method for determining relative specificities for sequential reactions catalyzed by the same enzyme: General formulation

被引:23
作者
Mitchell, David Alexander [1 ]
Carriere, Frederic [2 ]
Krieger, Nadia [3 ]
机构
[1] Univ Fed Parana, Dept Bioquim & Biol Mol, Ctr Politecn, BR-81531990 Curitiba, Parana, Brazil
[2] CNRS UPR 9025, Lab Enzymol Interfacile PhysiolLipolyse, Marseille, France
[3] Univ Fed Parana, Dept Quim, BR-81531990 Curitiba, Parana, Brazil
来源
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS | 2008年 / 1784卷 / 04期
关键词
specificity constant; repeated-attack; sequential hydrolysis; enzyme kinetics; mathematical model; hydrolase;
D O I
10.1016/j.bbapap.2008.01.015
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
We present a general formulation of a model that can be used to analyze reaction profiles in systems in which a single enzyme catalyzes several sequential reactions with the same molecular backbone. The analysis of these so-called "repeated-attack systems" allows estimation of the specificities that the enzyme has for the various intermediate substrates that appear in the reaction mixture, relative to the specificity that it has for the initial substrate. Our analytical method has the important advantage that it is not affected by competitive or uncompetitive inhibition, nor by denaturation of the enzyme during the reaction. We carry out case studies in three different systems, the lipase-catalyzed alcoholysis of triacylglycerols, the phytasecatalyzed removal of phosphate groups from phytic acid and the -amylasc-catalyzed removal of maltose units from maltolieptaose. Our model fits well to all reaction profiles in which the phenomenon of processivity does not occur. It can therefore be used as a general toot for characterizing the relative specificities of "repeated-attack enzymes". (c) 2008 Elsevier B.V All rights reserved.
引用
收藏
页码:705 / 715
页数:11
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