Identification of Cys139 and Glu207 As Catalytically Important Groups in the Active Site of Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

• Published in: Biochemistry (Easton) Vol. 33; no. 14; pp. 4212 - 4217
• Main Authors: Street, Ian P, Coffman, Hazel R, Poulter, C. Dale, Baker, Jonathan A
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 12-04-1994
• Subjects: ACIDE GLUTAMIQUE; ACIDO GLUTAMICO; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Carbon-Carbon Double Bond Isomerases; Catalysis; CISTEINA; Claviceps purpurea; CYSTEINE; Cysteine - genetics; Cysteine - metabolism; DNA Primers; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glutamates - genetics; Glutamates - metabolism; Glutamic Acid; ISOMERASAS; ISOMERASE; Isomerases; Isomerases - genetics; Isomerases - metabolism; Molecular Sequence Data; MUTACION; Mutagenesis, Site-Directed; MUTATION; SACCHAROMYCES CEREVISIAE; Glutamic acid; Cysteine; Isomerases; Intramolecular oxidoreductases; Enzymatic activity; Structure activity relation; Enzyme; Active site; Site directed mutagenesis; Isopentenyl-diphosphate Δ-isomerase; Kinetic parameter
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00180a014

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., and 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