Functional roles of the N-terminal amino acid residues in the Mn(II)-L-malate binding and subunit interactions of pigeon liver malic enzyme

被引:14
作者
Chou, WY [1 ]
Huang, SM [1 ]
Chang, GG [1 ]
机构
[1] Natl Def Med Ctr, Dept Biochem, Taipei 100, Taiwan
来源
PROTEIN ENGINEERING | 1997年 / 10卷 / 10期
关键词
malic enzyme; N-terminal amino acid; quaternary structure; site-specific mutagenesis; substrate binding;
D O I
10.1093/protein/10.10.1205
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met-Lys-Lys-Gly-Tyr-Glu-. In this work, various mutants of the enzyme with individual or combinational deletion (Delta) or substitution at these amino acids were constructed and functionally expressed in Escherichia coli cells, A major protein band corresponding to an M-r of similar to 65 000 was observed for all recombinant enzymes in sodium dodecyl sulfate polyacrylamide gel electrophoresis, However, when examining by polyacrylamide gel electrophoresis under native conditions, the recombinant enzymes were found to possess a tetrameric structure with M-r similar to 260 000 or a mixture of tetramers and dimers with the exception of Delta(K2K3G4) and Delta(1-16) mutants, which existed exclusively as dimers at the protein concentration we employed, K3A and K3E also dissociated substantially, K(2,3)A was a tetramer but K(2,3)E essentially existed as dimers, All tetramers and dimers were enzymatically active in the gels, All mutants displayed a similar apparent K-m value for NADP(+), The apparent K-m for L-malate and Mn(II), on the other hand, was increased by 4-27-fold for the Delta(K2/K3) and the Delta(1-16) mutants, The small binding affinity of Delta(K2/K3) with Mn(II)-L-malate was specific. With additional deletion at positions 3 and/or 4, the Delta(K2K3), Delta(K2G4/K3G4) or Delta(K2K3G4) mutants exhibited similar kinetic properties for the wild type, The lysine residues at the positions 2 or 3 seem to be crucial for the correct active site conformation, The results indicate that the N-terminus of malic enzyme is located at the Mn(II)-L-malate binding domain of the active center and is also near the subunit's interface, These results were interpreted with our asymmetric double-dimer model for the enzyme in which the N-terminus was involved in the head-to-tail monomer-monomer interactions but not the dimer-dimer interactions.
引用
收藏
页码:1205 / 1211
页数:7
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