Strain Is More Important Than Electrostatic Interaction in Conrolling the pK_a of the Catalytic Group in Aspartate Aminotransferase

摘要

Systematic single and multiple replacement studies have been applied to Escherichia coli aspartate 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 pKa value of the Schiff base formed between pyridoxal5'-phosphate (PLP) and Lys258. The electrostatic effects of the two arginine residues cannot be assessed by simple mutational studies of the residues. PLP-N-R lowers the pKa value of the PLP-Lys258 Schiff base by keeping it in the distorted conformation, which is unfavorable for protonation. Mutation of Arg386 ~liminates its hydrogen bond with Asn194 and partially disrupts PLP-N-R, thereby relaxing the strain of the Schiff base. On the other hand, mutation of Arg292, the large domain residue that interacts with the small domain residue Asp15, makes the domain opening easier. Because PLP-N-R lies between the two :lomains, the domain opening increases the strain of the Schiff base. Therefore, the true electrostatic ~ffects 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 dissect the electrostatic and structural effects of the arginine mutations on the Schiff base pKa. The positive ::harges of the two arginine residues and the PLP-N-R-mediated strain of the Schiff base lower the Schiff )ase pKa by 0.7 and 1.7, respectively. Thus, the electrostatic effect of the arginine residues is not as ;trong as has historically been thought, and this finding substantiates our recent finding that the imine- )yridine torsion of the Schiff base is the primary determinant (2.8 unit decrease) of the extremely low )Ka value of the Schiff base [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37, 15076-150851,