Abstract
Squalene synthetase (SQS, EC 2.5.1.21) catalyzes the first committed step in the formation of cholesterol and thus represents an ideal site for selectively inhibiting sterol formation. Previous studies have demonstrated that the fungal metabolite, zaragozic acid A (ZGA-A), inhibits SQS activity by mimicking the substrate farnesyl pyrophosphate, the reaction intermediate presqualene pyrophosphate, or both, through a process that confers increased apparent potency in the presence of reduced enzyme concentrations, an observation consistent with either tight binding reversible competitive inhibition or mechanism-based irreversible inactivation. The studies outlined in this report provide multiple lines of evidence indicating that ZGA-A acts as a mechanism-based irreversible inactivator of SQS. 1) Inhibition of SQS by ZGA-A is dependent on the [SQS] present in the incubation reaction, and this inhibition is time-dependent and follows pseudo-first order reaction kinetics, exhibiting kobs values that range between 2 x 10(-4)/s and 23 x 10(-4)/s for [ZGA-A] within the log-linear range of the inhibition curve, and a bimolecular rate constant of 2.3 x 10(5) M-1s-1.2) SQS activity is titratable by ZGA-A, such that for each [ZGA-A] evaluated, inactivation exhibits a threshold [SQS] whereby enzyme activity at lower [SQS] is totally inhibited. 3) Time-dependent inactivation exhibits saturation kinetics with a Km for the process of 2.5 nM, which is approximately equal to the IC50 for SQS inhibition under these conditions, suggesting that inactivation results from selective modification of a functional group of the enzyme active center rather than from a nonspecific bimolecular reaction mechanism and that most, if not all of the inhibition results from irreversible inactivation. 4) Saturable, time-dependent inactivation occurs with similar inactivation kinetics for both the microsomal and trypsin-solubilized forms of the enzyme, indicating that irreversible inactivation by ZGA-A is not a consequence of membrane modification but is a direct effect of the inhibitor on the enzyme. 5) Inactivation is biphasic, exhibiting a rapid ("burst") phase followed by a second, pseudo-first order phase, similar to that previously noted for irreversible inactivators in other enzyme systems, and occurs even in the presence of 5 mM concentrations of the nucleophylic scavenger dithiothreitol, suggesting that the reaction between ZGA-A and SQS occurs at or near the active center prior to diffusion of reactive species out of the catalytic cleft. 6) Inactivation can be prevented through competition with the substrate, farnesyl pyrophosphate, further identifying the active center as the site of modification.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
Squalene synthetase (SQS)1, a 47-kDa integral membrane protein of the endoplasmic reticulum 0), catalyzes the head-to-head condensation of two molecules of farnesyl pyrophosphate (FPP) to form squalene [1,2,3,4]
Based on these and other studies, plausible reaction mechanisms for the conversion of FPP to PSQPP and for the conversion of PSQPP to squalene have been advanced 0-3, 6, 11). Based on these studies, it has been postulated that the SQS catalytic machinery consists of two nonidentical FPP binding sites [3, 4, 24, 26] with different substrate [3, 4, 24, 26] and inhibitor [33] binding affinities, one site that binds FPP in a conformation that facilitates its heterolytic cleavage to yield an allylic carbocation that acts as a farnesyl donor [1,2,3], and one site that binds FPP in an orientation that facilitates insertion of the developing carbocation from the donor FPP residue into the C-1-C-2 double bond of the acceptor FPP to form the cyclopropylcarbinyl diphosphate intermediate, PSQPP [1,2,3]
Time-dependent Inactivation of SQS by zaragozic acid A (ZGA-A)-While not consistent with simple classical competitive inhibition, the observation that SQS inhibition by ZGA-A is dependent on the concentration of microsomal protein present in the incubation reaction is consistent with either 1) tight binding reversible inhibition, where the concentration of inhibitor is not in great excess relative to the concentration of enzyme and E· I complex formation significantly reduces the concentration of free inhibitor present at steady state to produce declining inhibition with increasing enzyme concentrations [57,58,59] or 2) mechanismbased irreversible inactivation where enzyme-inhibitor association reduces free inhibitor concentration through formation of nondisassociable E· I complexes, and reduces the concentration of catalytically active enzyme to produce greater inhibition at reduced enzyme concentrations
Summary
Vol 270, No 16, Issue of April 21, pp, 9083-9096, 1995 Printed in U.S.A. Inhibition of Mammalian Squalene Synthetase Activity by Zaragozic Acid A Is a Result of Competitive Inhibition Followed by Mechanism-based Irreversible Inactivation*. Inhibitors, that would interfere with various aspects of the catalytic reactions leading to the formation of PSQPP from FPP or to the formation of squalene from PSQPP could serve to further our understanding of the unique reaction mechanisms catalyzed by this enzyme but could serve to further our understanding of the regulatory mechanisms controlling mevalonate metabolism and the differential utilization of FPP by the various prenyltransferases catalyzing key reactions in the pathways leading to the sterols and nonsterol polyisoprenoids In this regard, a variety of studies using either FPP analogs as alternative substrates [3, 4, 24,25,26] or reaction inhibitors that act either as ground state mimics of the substrate FPP [27,28,29,30,31,32,33], ground state mimics of the reaction intermediate PSQPP [4, 13], or mimics ofthe putative carbocationic transition state intermediates of either the first or second half-reactions [1, 7,8,9,10,11,12] have been designed to probe the substrate binding sites, reaction centers, and catalytic mechanisms of the two half-reactions catalyzed by SQS. ZGA-A and related analogs may serve as useful tools to further probe the substrate binding sites and catalytic reaction centers within the active center of SQS and to further evaluate the unique catalytic mechanisms of this important enzyme
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