Strain Is More Important Than Electrostatic Interaction in Controlling the pKa of the Catalytic Group in Aspartate Aminotransferase

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• 作者：Hiroyuki Mizuguchi ; Hideyuki Hayashi ; Kengo Okada ; Ikuko Miyahara ; Ken Hirotsu ; and Hiroyuki Kagamiyama
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：January 16, 2001
• 年：2001
• 卷：40
• 期：2
• 页码：353 - 360
• 全文大小：618K
• 年卷期：v.40,no.2(January 16, 2001)
• ISSN：1520-4995

文摘

Systematic single and multiple replacement studies have been applied to Escherichia coliaspartate aminotransferase to probe the electrostatic effect of the two substrate-binding arginine residues,Arg292 and Arg386, and the structural effect of the pyridoxal 5'-phosphate-Asn194-Arg386 hydrogen-bond linkage system (PLP-N-R) on the pK_a value of the Schiff base formed between pyridoxal 5'-phosphate(PLP) and Lys258. The electrostatic effects of the two arginine residues cannot be assessed by simplemutational studies of the residues. PLP-N-R lowers the pK_a value of the PLP-Lys258 Schiff base bykeeping it in the distorted conformation, which is unfavorable for protonation. Mutation of Arg386eliminates its hydrogen bond with Asn194 and partially disrupts PLP-N-R, thereby relaxing the strain ofthe Schiff base. On the other hand, mutation of Arg292, the large domain residue that interacts with thesmall domain residue Asp15, makes the domain opening easier. Because PLP-N-R lies between the twodomains, the domain opening increases the strain of the Schiff base. Therefore, the true electrostaticeffects of Arg292 and Arg386 could be derived from mutational analysis of the enzyme in which PLP-N-R had been completely disrupted by the Asn194Ala mutation. Through the analyses, we could dissectthe electrostatic and structural effects of the arginine mutations on the Schiff base pK_a. The positivecharges of the two arginine residues and the PLP-N-R-mediated strain of the Schiff base lower the Schiffbase pK_a by 0.7 and 1.7, respectively. Thus, the electrostatic effect of the arginine residues is not asstrong as has historically been thought, and this finding substantiates our recent finding that the imine-pyridine torsion of the Schiff base is the primary determinant (2.8 unit decrease) of the extremely lowpK_a value of the Schiff base [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37,15076-15085].