J. Biol. Chem., Vol. 269, Issue 15, 11201-11207, Apr, 1994
Yeast squalene synthase. A mechanism for addition of substrates and activation by NADPH
KA Mookhtiar, SS Kalinowski, D Zhang and CD Poulter
Department of Metabolic Diseases, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543.
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.
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