Mizuguchi H, Hayashi H, Okada K, Miyahara I, Hirotsu K, Kagamiyama H
Department of Biochemistry, Osaka Medical College, Takatsuki 569-8686, Japan.
Biochemistry. 2001 Jan 16;40(2):353-60. doi: 10.1021/bi001403e.
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 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 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 protonation. Mutation of Arg386 eliminates 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 domains, the domain opening increases the strain of the Schiff base. Therefore, the true electrostatic effects 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 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 electrostatic effect of the arginine residues is not as strong 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 low pK(a) value of the Schiff base [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37, 15076-15085].
系统性的单取代和多取代研究已应用于大肠杆菌天冬氨酸转氨酶，以探究两个底物结合精氨酸残基（Arg292和Arg386）的静电效应，以及磷酸吡哆醛-天冬酰胺194-精氨酸386氢键连接系统（PLP-N-R）对磷酸吡哆醛（PLP）与赖氨酸258之间形成的席夫碱pK(a)值的结构效应。两个精氨酸残基的静电效应无法通过对这些残基进行简单的突变研究来评估。PLP-N-R通过使PLP-赖氨酸258席夫碱保持扭曲构象而降低其pK(a)值，这种构象不利于质子化。精氨酸386的突变消除了它与天冬酰胺194的氢键，并部分破坏了PLP-N-R，从而缓解了席夫碱的张力。另一方面，与小结构域残基天冬氨酸15相互作用的大结构域残基精氨酸292的突变，使结构域打开更容易。由于PLP-N-R位于两个结构域之间，结构域打开增加了席夫碱的张力。因此，精氨酸292和精氨酸386的真正静电效应可以从对天冬酰胺194丙氨酸突变完全破坏了PLP-N-R的酶的突变分析中得出。通过这些分析，我们可以剖析精氨酸突变对席夫碱pK(a)的静电和结构效应。两个精氨酸残基的正电荷以及PLP-N-R介导的席夫碱张力分别使席夫碱pK(a)降低0.7和1.7。因此，精氨酸残基的静电效应并不像以往认为的那么强，这一发现证实了我们最近的发现，即席夫碱的亚胺-吡啶扭转是席夫碱极低pK(a)值的主要决定因素（降低2.8个单位）[林，H.，水口，H.，和加贺山，H.（1998年）《生物化学》37，15076 - 15085]。
