Yeast squalene synthase. A mechanism for addition of substrates and activation by NADPH

K.A Mookhtiar,S.S Kalinowski,D Zhang,C.D Poulter

doi:10.1016/s0021-9258(19)78111-3

K.A Mookhtiar, S.S Kalinowski + Show 2 more

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https://doi.org/10.1016/s0021-9258(19)78111-3

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Abstract

Squalene synthase catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the subsequent reductive rearrangement of PSPP to squalene. Previous studies of the mechanism of addition of FPP to the enzyme have led to conflicting interpretations of initial velocity measurements (Beytia, E., Qureshi, A. A., and Porter, J.W. (1973) J. Biol. Chem. 248, 1856-1867; Agnew, W.S., and Popjak, G. (1978) J. Biol. Chem. 253, 4566-4573). Initial velocities for synthesis of PSPP and squalene were measured over a wider range of FPP and NADPH concentrations than previously reported, using a soluble form of recombinant enzyme. In the absence of NADPH, PSPP formation was activated by FPP at concentrations above approximately 0.5 microM. At fixed levels of NADPH, the dependence of initial rates of PSPP and squalene synthesis on FPP concentrations indicated that the C15 substrate added by a sequential mechanism. In addition, NADPH stimulated synthesis of PSPP by 40-fold at saturating levels of the cofactor. This stimulation is, at least in part, by reduction of PSPP to squalene.

Highlights

From the Wepartment of Metabolic Diseases, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543 and the Wepartment of Chemistry, University of Utah, Salt Lake City, Utah 84112
This work was reported before studies of the mechanism of additionof FPP to the en- PSPP was discovered and did not take into account the more zyme have led to conflicting interpretations of initial velocity measurements
Initial Velocity Studies difference could result from a systematic error in correlating the assays for PSPP and squalene.Within the error of our PSPP Synthesis in the Absence of NmPH-The initial ve- experiments, both compounds were formed at essentially idenlocity ( u ) for PSPP synthesis was measured at different FPP tical rates in buffer containing 1m~ NADPH where 0.1 p 5 concentrations in theabsence of NADPH by the tritium release [FPP] I 10 p.Similar behavior was reported by Agnew and assay

Summary

Introduction

From the Wepartment of Metabolic Diseases, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543 and the Wepartment of Chemistry, University of Utah, Salt Lake City, Utah 84112. Concentrationtshapnreviouslryeported, using a soluble form of recombinant enzyme.In the absence of NADPH, PSPP formation wasactivated by FPP at concentrations above”0.5 p ~At. fixed levels of NADPH,the dependence of initial rates of PSPP and squalene synthesis on FPP concentrations indicated thtahte c,,substrate added by a sequential mechanism.

Results

Conclusion