title: Mechanism of action of isopentenyl pyrophosphate isomerase: evidence for a carbonium ion intermediate.
abstract: Isopentenyl pyrophosphate isomerase catalyzes the interconversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. The isomerase from yeast has been purified to near homogeneity (purity greater than 90%). The substrate analogue (Z)-3-(trifluoromethyl)-2-butenyl pyrophosphate reacts at less than 1.8 X 10(-6) times the rate of dimethylallyl pyrophosphate. The enzyme is irreversibly inactivated by 2-(dimethyl-amino)ethyl pyrophosphate (I). These observations are consistent with a carbonium ion mechanism for the isomerization. Compound I is an analogue of the intermediate carbonium ion and probably acts as a transition state analogue. For I, kon' = 2.1 X 10(6) M-1 min-1. No off-rate was detected and, therefore, Ki less than 1.4 X 10(-11) M. Upon denaturation of the inactivated enzyme, I is released unchanged. 2-(Trimethylammonio)ethyl pyrophosphate also inhibits with Ki' = 7 X 10(-7) M, kon' = 4.4 X 10(4) M-1 min-1, and koff = 0.03 min-1. Substrate analogues without a positively charged nitrogen were relatively poor inhibitors. The best inhibitor of these is ethyl pyrophosphate, Ki = 10(-4) M. The enzyme is inactivated by sulfhydryl-selective reagents. These reagents also prevent binding of I to the enzyme. The inactivation by iodoacetamide is dependent upon one ionizable group (pK = 9.3). The pH dependence of V and V/K for the isomerase-catalyzed reaction also depends upon a group with pK = 9.3.
mesh_terms: D019748:Carbon-Carbon Double Bond Isomerases; D011756:Diphosphates / Q000494:pharmacology; D045782:Hemiterpenes; D007535:Isomerases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D011756:Diphosphates; D045782:Hemiterpenes; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase