Accurate determination of rate constants of very slow, tight-binding competitive inhibitors by numerical solution of differential equations, independently of precise knowledge of the enzyme concentration

被引:5
作者
Plesner, IW [1 ]
Bülow, A [1 ]
Bols, M [1 ]
机构
[1] Aarhus Univ, Dept Chem, DK-8000 Aarhus C, Denmark
关键词
transition state kinetics; enzymes; competitive inhibitors; slow binding; differential equations; rate constant determination; beta-glucosidase; thermodynamic functions of binding;
D O I
10.1006/abio.2001.5221
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
This paper is concerned with the determination of rate constants characterizing the binding and release of a slow binding inhibitor to and from an enzyme, here almond beta -glucosidase. We demonstrate the inability of the conventional method to yield reliable rate constants when one or more of these is less than 1 x 10(-4) per second. Instead one must use the much more accurate fitting of rate constants of the set of simultaneous differential equations characterizing the kinetic model. This procedure has the added advantage, when properly used, that the rate constants found pertaining to the inhibitor are largely insensitive to the particular value used for the enzyme concentration; i.e., the same data set may be fitted using a range of enzyme concentrations with no change in the resulting parameters. Hence the method can be used when little is known about the enzyme, except for the value of Km, which is readily determined. Also, we report the somewhat unexpected finding that the association rate constant for the substrate (4-nitrophenyl-beta -D-glucopyranoside) is about one-third of the value of the corresponding rate constant for the inhibitor. The method is used to determine rate constants at several temperatures for the strong, slow binding inhibitor 2-phenethylglucoimidazole 1, enabling us to compute standard thermodynamic functions. The identity of these functions with those of isofagomine (2) reported earlier leads us to argue that the two compounds share a common binding mechanism, involving the same groups, whereas the different stabilities of the enzyme-inhibitor complexes must reside in those parts of the molecules that are not identical. (C) 2001 Academic Press.
引用
收藏
页码:186 / 193
页数:8
相关论文
共 15 条
[1]   A large difference in the thermodynamics of binding of isofagomine and 1-deosynojirimycin to β-glucosidase [J].
Bülow, A ;
Plesner, IW ;
Bols, M .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2000, 122 (35) :8567-8568
[2]   Slow inhibition of almond β-glucosidase by azasugars:: determination of activation energies for slow binding [J].
Bülow, A ;
Plesner, IW ;
Bols, M .
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 2001, 1545 (1-2) :207-215
[3]  
Ernholt BV, 2000, CHEM-EUR J, V6, P278, DOI 10.1002/(SICI)1521-3765(20000117)6:2<278::AID-CHEM278>3.0.CO
[4]  
2-6
[5]  
Hansen SU, 2000, CHEMBIOCHEM, V1, P177
[6]  
Heightman TD, 1999, ANGEW CHEM INT EDIT, V38, P750, DOI 10.1002/(SICI)1521-3773(19990315)38:6<750::AID-ANIE750>3.0.CO
[7]  
2-6
[8]   General numerical treatment of competitive binding kinetics: Application to thrombin-dehydrothrombin-hirudin [J].
Kuzmic, P .
ANALYTICAL BIOCHEMISTRY, 1999, 267 (01) :17-23
[9]   Investigation of the slow inhibition of almond β-glucosidase and yeast isomaltase by 1-azasugar inhibitors:: evidence for the 'direct binding' model [J].
Lohse, A ;
Hardlei, T ;
Jensen, A ;
Plesner, IW ;
Bols, M .
BIOCHEMICAL JOURNAL, 2000, 349 :211-215