Acceleration of the substrate Cα deprotonation by an analogue of the second substrate palmitoyl-CoA in serine palmitoyltransferase

Abstract

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the pyridoxal 5′-phosphate (PLP)-dependent decarboxylative condensation reaction of L-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The binding of L-serine alone to SPT leads to the formation of the external aldimine but does not produce a detectable amount of the quinonoid intermediate. However, the further addition of S-(2-oxoheptadecyl)-CoA, a nonreactive analogue of palmitoyl-CoA, caused the apparent accumulation of the quinonoid. NMR studies showed that the hydrogen-deuterium exchange at Cα of L-serine is very slow in the SPT-L-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. In addition, the observation that the nonreactive palmitoyl-CoA facilitated α-deprotonation indicates that the α-deprotonation takes place before the Claisen-type C-C bond formation, which is consistent with the accepted mechanism of the α-oxamine synthase subfamily enzymes. Structural modeling of both the SPT-L-serine external aldimine complex and SPT-L-serine-palmitoyl-CoA ternary complex suggests a mechanism in which the binding of palmitoyl-CoA to SPT induced a conformation change in the PLP-L-serine external aldimine so that the Cα-H bond of L-serine becomes perpendicular to the plane of the PLP-pyridine ring and is favorable for the α-deprotonation. The model also proposed that the two alternative hydrogen bonding interactions of His159 with L-serine and palmitoyl-CoA play an important role in the conformational change of the external aldimine. This is the unique mechanism of SPT that prevents the formation of the reactive intermediate before the binding of the second substrate. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.