Identification of Cys139 and Glu207 as catalytically important groups in the active site of isopentenyl diphosphate:dimethylallyl diphosphate isomerase.
Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (EC 5.3.3.2) catalyzes the antarafacial [1.3] allylic rearrangement of isopentenyl diphosphate (IPP) to its electrophilic allylic isomer dimethylallyl diphosphate (DMAPP). Active-site thiols at C138 and C139 were recently identified by covalent modification using active-site-directed irreversible inhibitors [Street, I. P., & Poulter, C. D. (1990) Biochemistry 29, 7531-7538; Lu, X. J., Christensen, D. J., & Poulter, C. D. (1992) Biochemistry 31, 9955-9960]. Kinetic studies were conducted with site-directed mutants of IPP isomerase (IPPIase) to evaluate the roles of these amino acids. C138S and C138V mutants were active catalysts with V/K values only 10-fold lower than that of wild-type IPPIase. In contrast, the C139S mutant was a poor catalyst, and the C139A and C139V mutants were inactive. Treatment of the C139S mutant with 3-(fluoromethyl)-3-butenyl diphosphate, an electrophilic active-site-directed irreversible inhibitor, resulted in inactivation of the enzyme by covalent modification of E207. The E207Q and E207V mutants were inactive, suggesting a role for the E207 carboxylate moiety in catalysis.
Street
I P
IP
Department of Chemistry, University of Utah, Salt Lake City 84112.
Coffman
H R
HR
Baker
J A
JA
Poulter
C D
CD
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D019748:Carbon-Carbon Double Bond Isomerases; D002384:Catalysis; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D005971:Glutamates / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid; D045782:Hemiterpenes; D007535:Isomerases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D017931:DNA Primers; D005971:Glutamates; D045782:Hemiterpenes; D018698:Glutamic Acid; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase; D003545:Cysteine