Isolation and characterization of mycophenolic acid-resistant mutants of inosine-5'-monophosphate dehydrogenase

被引:42
作者
Farazi, T [1 ]
Leichman, J [1 ]
Harris, T [1 ]
Cahoon, M [1 ]
Hedstrom, L [1 ]
机构
[1] BRANDEIS UNIV, GRAD DEPT BIOCHEM, WALTHAM, MA 02254 USA
关键词
D O I
10.1074/jbc.272.2.961
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
Mycophenolic acid (MPA) is a potent and specific inhibitor of mammalian inosine-monophosphate dehydrogenases (IMPDH); most microbial IMPDHs are not sensitive to MPA. MPA-resistant mutants of human IMPDH type II were isolated in order to identify the structural features that determine the species selectivity of MPA, Three mutant IMPDHs were identified with decreased affinity for MPA. The mutation of Gln(277) --> Arg causes a 9-fold increase in the K-i of MPA, a 5-6 fold increase in the K-m values for IMP and NAD, and a 3-fold decrease in k(cat) relative to wild type, The mutation of Ala(462) --> Thr causes a 3-fold increase in the K-i for MPA, a 2.5-fold increase in the K-m for NAD, and a 1.5 fold increase in k(cat). The combination of these two mutations does not increase the K-i for MPA, but does increase the K-m for NAD 3-fold relative to Q277R and restores k(cat) to wild type levels, Q277R/A462T is the first human IMPDH mutant with increased K-i for MPA and wild type activity. The third mutant IMPDH contains two mutations, Phe(465) --> Ser and Asp(470) --> Gly. K-i for MPA is increased 3-fold in this mutant enzyme, and K-m for IMP is also increased 3-fold, while the K-m for NAD and k(cat) are unchanged, Thus increases in the K-i for MPA do not correlate with changes in K-m for either IMP or NAD, nor to changes in k(cat). All four of these mutations are in regions of the IMPDH that differ in mammalian and microbial enzymes, and thus can be structural determinants of MPA selectivity.
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页码:961 / 965
页数:5
相关论文
共 44 条
[11]   Inhibition of IMPDH by mycophenolic acid: Dissection of forward and reverse pathways using capillary electrophoresis [J].
Fleming, MA ;
Chambers, SP ;
Connelly, PR ;
Nimmesgern, E ;
Fox, T ;
Bruzzese, FJ ;
Hoe, ST ;
Fulghum, JR ;
Livingston, DJ ;
Stuver, CM ;
Sintchak, MD ;
Wilson, KP ;
Thomson, JA .
BIOCHEMISTRY, 1996, 35 (22) :6990-6997
[12]  
Franklin TJ, 1996, CANCER RES, V56, P984
[13]   INHIBITION OF NUCLEIC ACID SYNTHESIS BY MYCOPHENOLIC ACID [J].
FRANKLIN, TJ ;
COOK, JM .
BIOCHEMICAL JOURNAL, 1969, 113 (03) :515-+
[14]   CLONING AND SEQUENCE OF THE HUMAN TYPE-II IMP DEHYDROGENASE GENE [J].
GLESNE, DA ;
HUBERMAN, E .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1994, 205 (01) :537-544
[15]  
GRECO WR, 1979, J BIOL CHEM, V254, P2104
[16]   RECOMBINANT HUMAN INOSINE MONOPHOSPHATE DEHYDROGENASE TYPE-I AND TYPE-II PROTEINS - PURIFICATION AND CHARACTERIZATION OF INHIBITOR BINDING [J].
HAGER, PW ;
COLLART, FR ;
HUBERMAN, E ;
MITCHELL, BS .
BIOCHEMICAL PHARMACOLOGY, 1995, 49 (09) :1323-1329
[17]   A NOVEL MECHANISM OF MYCOPHENOLIC-ACID RESISTANCE IN THE PROTOZOAN PARASITE TRITRICHOMONAS-FETUS [J].
HEDSTROM, L ;
CHEUNG, KS ;
WANG, CC .
BIOCHEMICAL PHARMACOLOGY, 1990, 39 (01) :151-160
[18]   CONVERTING TRYPSIN TO CHYMOTRYPSIN - THE ROLE OF SURFACE LOOPS [J].
HEDSTROM, L ;
SZILAGYI, L ;
RUTTER, WJ .
SCIENCE, 1992, 255 (5049) :1249-1253
[19]   MYCOPHENOLIC-ACID AND THIAZOLE ADENINE-DINUCLEOTIDE INHIBITION OF TRITRICHOMONAS-FETUS INOSINE 5'-MONOPHOSPHATE DEHYDROGENASE - IMPLICATIONS ON ENZYME MECHANISM [J].
HEDSTROM, L ;
WANG, CC .
BIOCHEMISTRY, 1990, 29 (04) :849-854
[20]   IDENTIFICATION OF THE IMP BINDING-SITE IN THE IMP DEHYDROGENASE FROM TRITRICHOMONAS-FETUS [J].
HUETEPEREZ, JA ;
WU, JC ;
WHITBY, FG ;
WANG, CC .
BIOCHEMISTRY, 1995, 34 (42) :13889-13894