Characterization of recombinant guinea pig alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli: Kinetics, chemical modification and mutagenesis

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• 作者：de Vet ; Edwin C.J.M. ; van den Bosch ; Henk
• 关键词：Ether lipid synthesis ; Alkyl-dihydroxyacetonephosphate synthase ; Mechanism ; Site-directed mutagenesis ; Essential residue ; DHAP ; dihydroxyacetonephosphate synthase ; DNFB ; 2 ; 4-dinitrofluorobenzene ; NEM ; N-ethylmaleimide ; pBPB ; p-bromophenacylbromide
• 刊名：Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids
• 出版年：1999
• 出版时间：January 4, 1999
• 年：1999
• 卷：1436
• 期：3
• 页码：299-306
• 全文大小：278 K

文摘

A recombinant form of guinea pig alkyl-dihydroxyacetonephosphate synthase, a key enzyme in the biosynthesis of ether phospholipids, was characterized. Kinetic analysis yielded evidence that the enzyme operates by a ping-pong rather than a sequential mechanism. Enzyme activity was irreversibly inhibited by N-ethylmaleimide, p-bromophenacylbromide and 2,4-dinitrofluorobenzene. The enzyme could be protected against the inactivation by either of these three compounds by the presence of saturating amounts of the substrate palmitoyl-dihydroxyacetonephosphate. The rate of inactivation of the enzyme by p-bromophenacylbromide was strongly pH dependent and the highest at alkaline conditions. Collectively, these results are indicative of cysteine, histidine and lysine residues, respectively, at or close to the active site. The divalent cations Mg²⁺, Zn²⁺ and Mn²⁺ were found to be inhibitors of enzymatic activity, whereas Ca²⁺ had no effect. Mutational analysis showed that histidine 617 is an essential amino acid for enzymatic activity: replacement of this residue by alanine resulted in complete loss of enzymatic activity. A recombinant enzyme with the C-terminal five amino acids deleted was shown to be inactive, indicating an important role of the C-terminus for catalytic activity.