DIHYDROFOLATE-REDUCTASE FROM ESCHERICHIA-COLI - PROBING THE ROLE OF ASPARTATE-27 AND PHENYLALANINE-137 IN ENZYME CONFORMATION AND THE BINDING OF NADPH

被引:24
作者
DUNN, SMJ
LANIGAN, TM
HOWELL, EE
机构
[1] UNIV TENNESSEE,DEPT BIOCHEM,KNOXVILLE,TN 37796
[2] UNIV IOWA,DEPT PHYSIOL & BIOPHYS,IOWA CITY,IA 52242
关键词
D O I
10.1021/bi00489a010
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
In the absence of ligands, dihydrofolate reductase from Escherichia coli exists in at least two interconvertible conformations, only one of which binds NADPH with high affinity. This equilibrium is pH dependent, involving an ionizable group of the enzyme (pK ~ 5.5), and the proportion of the NADPH-binding conformer increases from 42% at pH 5 to 65% at pH 8. The role of specific amino acids in enzyme conformation has been investigated by studying the kinetics of NADPH binding to three dihydrofolate reductase mutants: (i) a mutant in which Asp-27, a residue that is directly involved in the binding of folates and antifolates but not NADPH, has been replaced by a serine, (ii) a mutant in which Phe-137 on the exterior of the molecule and distant from the binding sites has been replaced by a serine, and (iii) a mutant in which both Asp-27 and Phe-137 have been replaced by serines. Mutation of the Asp-27 residue reduces the affinity for NADPH by approximately 7-fold. Kinetic measurements have suggested that this is due mainly to an increase in the rate of dissociation of the initial complex and a slight shift in the enzyme equilibrium to favor the nonbinding conformation. The pH dependence of the conformer equilibrium is also shifted by approximately one pH unit to higher pH (pK ~ 6.5). In addition, the pH profile suggests the involvement of a second ionizable group having a pK of about 8 since, above pH 7, the proportion of the NADPH-binding form decreases. Evidence for the involvement of a second ionizable group with a similar pK has been obtained for the Ser-137 mutant, but otherwise, the kinetics of NADPH binding to this enzyme are not significantly different from those of the wild type. Replacement of both Asp-27 and Phe-137 by serines resulted in an obvious change in the NADPH-binding kinetics. In contrast to the wild type, the proportion of the NADPH-binding form decreased from 54% to 42% between pH 5 and pH 8. This may be explained by changes in the equilibrium constants and pK values of the ionizable group(s) involved in the equilibrium between the two enzyme conformations.© 1990, American Chemical Society. All rights reserved.
引用
收藏
页码:8569 / 8576
页数:8
相关论文
共 32 条
[1]   EFFECTS OF DISTAL POINT-SITE MUTATIONS ON THE BINDING AND CATALYSIS OF DIHYDROFOLATE-REDUCTASE FROM ESCHERICHIA-COLI [J].
ADAMS, J ;
JOHNSON, K ;
MATTHEWS, R ;
BENKOVIC, SJ .
BIOCHEMISTRY, 1989, 28 (16) :6611-6618
[2]   A KINETIC-STUDY OF WILD-TYPE AND MUTANT DIHYDROFOLATE REDUCTASES FROM LACTOBACILLUS-CASEI [J].
ANDREWS, J ;
FIERKE, CA ;
BIRDSALL, B ;
OSTLER, G ;
FEENEY, J ;
ROBERTS, GCK ;
BENKOVIC, SJ .
BIOCHEMISTRY, 1989, 28 (14) :5743-5750
[3]  
APPLEMAN JR, 1990, J BIOL CHEM, V265, P5579
[4]   ANALYSIS OF NUMERICAL-METHODS FOR COMPUTER-SIMULATION OF KINETIC PROCESSES - DEVELOPMENT OF KINSIM - A FLEXIBLE, PORTABLE SYSTEM [J].
BARSHOP, BA ;
WRENN, RF ;
FRIEDEN, C .
ANALYTICAL BIOCHEMISTRY, 1983, 130 (01) :134-145
[5]  
BENKOVIC SJ, 1988, SCIENCE, V239, P1104
[6]  
BOLIN JT, 1982, J BIOL CHEM, V257, P13650
[7]   KINETICS OF SUBSTRATE, COENZYME, AND INHIBITOR BINDING TO ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE [J].
CAYLEY, PJ ;
DUNN, SMJ ;
KING, RW .
BIOCHEMISTRY, 1981, 20 (04) :874-879
[8]   SITE-SPECIFIC MUTAGENESIS OF DIHYDROFOLATE-REDUCTASE FROM ESCHERICHIA-COLI [J].
CHEN, JT ;
MAYER, RJ ;
FIERKE, CA ;
BENKOVIC, SJ .
JOURNAL OF CELLULAR BIOCHEMISTRY, 1985, 29 (02) :73-82
[9]   PROBING THE FUNCTIONAL-ROLE OF PHENYLALANINE-31 OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE BY SITE-DIRECTED MUTAGENESIS [J].
CHEN, JT ;
TAIRA, K ;
TU, CPD ;
BENKOVIC, SJ .
BIOCHEMISTRY, 1987, 26 (13) :4093-4100
[10]   KINETICS OF LIGAND-BINDING TO DIHYDROFOLATE-REDUCTASE - BINARY COMPLEX-FORMATION WITH NADPH AND COENZYME ANALOGS [J].
DUNN, SMJ ;
BATCHELOR, JG ;
KING, RW .
BIOCHEMISTRY, 1978, 17 (12) :2356-2364