IONIZATION STATE AND PK(A) OF PTERIN-ANALOG LIGANDS BOUND TO DIHYDROFOLATE-REDUCTASE

被引:12
作者
JEONG, SS [1 ]
GREADY, JE [1 ]
机构
[1] UNIV SYDNEY,DEPT BIOCHEM,SYDNEY,NSW 2006,AUSTRALIA
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1994年 / 221卷 / 03期
关键词
D O I
10.1111/j.1432-1033.1994.tb18824.x
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 ionization state and pK(a) of the inhibitor 6,8-dimethyl-N5-deazapterin bound to the recombinant human dihydrofolate reductase (rhH(2) folate reductase) complex with NADPH was determined by a spectrofluorimetric method. The excitation spectra for bound ligand as a function of pH from 6.1 to 9.7 indicated it was the same cationic form as for unbound ligand, which is protonated on N3. However, the lower limit for the pK(a) of the bound form was determined to be 9.1, a value about pH 2.5 higher than that for free Ligand, indicating that ligand bound to the enzyme is protonated at neutral pH. The excitation spectra for bound ligand as a function of pH were generated by computer simulation by employing corrections for the pH dependence of the proportion of bound ligand (variable K-d; ligand-dissociation constant) and taking account of the different pK(a) values for bound and unbound forms. A plot of K-d values against pH showed a bell-shaped curve indicating that 6,8-dimethyl-N5-deazapterin bound to rhH(2) folate reductase . NADPH to form a ternary complex of ionised enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand; the spectrofluorimetric results are consistent with the first alternative.
引用
收藏
页码:1055 / 1062
页数:8
相关论文
共 23 条
[1]   CORRECTION FOR LIGHT-ABSORPTION IN FLUORESCENCE STUDIES OF PROTEIN-LIGAND INTERACTIONS [J].
BIRDSALL, B ;
KING, RW ;
WHEELER, MR ;
LEWIS, CA ;
GOODE, SR ;
DUNLAP, RB ;
ROBERTS, GCK .
ANALYTICAL BIOCHEMISTRY, 1983, 132 (02) :353-361
[2]   C-13 NUCLEAR MAGNETIC-RESONANCE STUDY OF PROTONATION OF METHOTREXATE AND AMINOPTERIN BOUND TO DIHYDROFOLATE-REDUCTASE [J].
COCCO, L ;
GROFF, JP ;
TEMPLE, C ;
MONTGOMERY, JA ;
LONDON, RE ;
MATWIYOFF, NA ;
BLAKLEY, RL .
BIOCHEMISTRY, 1981, 20 (14) :3972-3978
[3]  
ELLIS KJ, 1982, METHOD ENZYMOL, V87, P405
[4]  
GREADY JE, 1990, CHEMISTRY AND BIOLOGY OF PTERIDINES 1989, P23
[5]  
GREADY JE, 1993, CHEM BIOL PTERIDINES, P487
[6]  
GREADY JE, 1992, PTERIDINES RELATED B, P265
[7]   ULTRAVIOLET DIFFERENCE-SPECTROSCOPIC STUDIES OF SUBSTRATE AND INHIBITOR BINDING TO LACTOBACILLUS-CASEI DIHYDROFOLATE-REDUCTASE [J].
HOOD, K ;
ROBERTS, GCK .
BIOCHEMICAL JOURNAL, 1978, 171 (02) :357-366
[8]   APPROACHES TO THE PREPARATION OF 6- AND 7-METHYL-8-SUBSTITUTED PTERINS .1. THE EFFECT OF REACTION CONDITIONS ON ISOMER DISTRIBUTION AND A NOVEL-APPROACH TO ISOMER SEPARATION [J].
IVERY, MTG ;
GREADY, JE .
BIOLOGICAL CHEMISTRY HOPPE-SEYLER, 1992, 373 (11) :1125-1137
[9]  
IVERY MTG, 1993, CHEM BIOL PTERIDINES, P525
[10]   PH-DEPENDENCE OF THE STRUCTURE AND DEGRADATION OF 8-ALKYL-SUBSTITUTED PTERINS [J].
JEONG, SS ;
GREADY, JE .
BIOLOGICAL CHEMISTRY HOPPE-SEYLER, 1992, 373 (11) :1139-1157