Strain is more important than electrostatic interaction in controlling thepK(a) of the catalytic group in aspartate aminotransferase

Systematic single and multiple replacement studies have been applied to Esc herichia 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 pK(a) value of the Schiff base formed betw een pyridoxal 5'-phosphate (PLP) and Lys258. The electrostatic effects of t he two arginine residues cannot be assessed by simple mutational studies of the residues, PLP-N-R lowers the pK(a) value of the PLP-Lys258 Schiff base by keeping it in the distorted conformation, which is unfavorable for prot onation. Mutation of Arg386 eliminates its hydrogen bond with Asn194 and pa rtially disrupts PLP-N-R, thereby relaxing the strain of the Schiff base. O n the other hand, mutation of Arg292, the large domain residue that interac ts with the small domain residue Asp15, makes the domain opening easier. Be cause PLP-N-R lies between the two domains, the domain opening increases th e strain of the Schiff base. Therefore, the true electrostatic effects of A rg292 and Arg386 could be derived from mutational analysis of the enzyme in which PLP-N-R had been completely disrupted by the Asn194Ala mutation. Thr ough the analyses, we could dissect the electrostatic and structural effect s of the arginine mutations on the Schiff base pK(a). The positive charges of the two arginine residues and the PLP-N-R-mediated strain of the Schiff base lower the Schiff base pK(a) by 0.7 and 1.7, respectively. Thus, the el ectrostatic effect of the arginine residues is not as strong as has histori cally 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 low pK(a) value of the Schiff base.