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J. Biol. Chem., Vol. 283, Issue 12, 7542-7553, March 21, 2008

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Acceleration of the Substrate C Deprotonation by an Analogue of the Second Substrate Palmitoyl-CoA in Serine Palmitoyltransferase^*

Hiroko Ikushiro¹, Shigeru Fujii, Yuka Shiraiwa, and Hideyuki Hayashi²

From the Department of Biochemistry, Osaka Medical College, 2-7 Daigakumachi, Takatsuki, Osaka 569-8686 and the Laboratory of Chemistry, Kansai Medical University, Hirakata, Osaka 573-1136, Japan

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 C 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 -deprotonation indicates that the -deprotonation takes place before the Claisen-type C–C bond formation, which is consistent with the accepted mechanism of the -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 C–H bond of L-serine becomes perpendicular to the plane of the PLP-pyridine ring and is favorable for the -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.

Received for publication, August 17, 2007 , and in revised form, December 26, 2007.

^* This work was supported by Grant-in-Aid for Encouragement of Young Scientists (B) 16770103 and Grant-in-Aid for Scientific Research (C) 18570114 (to H. I.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by Grant-in-Aid for Scientific Research (C) 16570125 (to H. H.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed. Tel.: 81-72-684-7291; Fax: 81-72-684-6516; E-mail: ikushiro{at}art.osaka-med.ac.jp. ² To whom correspondence may be addressed. E-mail: hayashi{at}art.osaka-med.ac.jp.

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