A pH-dependent stabilization of an active site loop observed from low and high ph crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 Å

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• 作者：Su ; Ying ; Yamashita ; Mason M. ; Greasley ; Samantha E. ; Mullen ; Christine A. ; Shim ; Jae Hoon ; et. al.
• 关键词：purine biosynthesis ; folate cofactors ; loop flexibility ; monomer-dimer association ; enzyme mechanism ; GarTfase ; glycinamide ribonucleotide transformylase ; 10fTHF ; 10-formyl tetrahydrofolate ; β ; -Gar ; β ; -glycinamide ribonucleotide ; f-β ; -Gar ; for
• 刊名：Journal of Molecular Biology
• 出版年：1998
• 出版时间：August 21, 1998
• 年：1998
• 卷：281
• 期：3
• 页码：485-499
• 全文大小：2.80 M

文摘

A mutation in the dimer interface of Escherichia coli glycinamide ribonucleotide transformylase (GarTfase) disrupts the observed pH-dependent association of the wild-type enzyme, but has no observable effect on the enzyme activity. Here, we assess whether a pH effect on the enzyme’s conformation is sufficient by itself to explain the pH-dependence of the GarTfase reaction. A pH-dependent conformational change is observed between two high-resolution crystal structures of the Glu70Ala mutant GarTfase at pH 3.5 (1.8 Å) and 7.5 (1.9 Å). Residues 110 to 131 in GarTfase undergo a transformation from a disordered loop at pH 3.5, where the enzyme is inactive, to an ordered loop-helix structure at pH 7.5, where the enzyme is active. The ordering of this flexible loop-helix has a direct effect on catalytic residues in the active site, binding of the folate cofactor and shielding of the active site from solvent. A main-chain carbonyl oxygen atom from Tyr115 in the ordered loop forms a hydrogen bond with His108, and thereby provides electronic and structural stabilization of this key active site residue. Kinetic data indicate that the p K_aof His108 is in fact raised to 9.2. The loop movement can be correlated with elevation of the His p K_a, but with further stabilization, probably from Asp144, after the binding of folate cofactor. Leu118, also in the loop, becomes positioned near the p-amino benzoic acid binding site, providing additional hydrophobic interactions with the cofactor 10-formyl tetrahydrofolate. Thus, the pH-dependence of the enzyme activity appears to arise from local active site rearrangements and not from differences due to monomer-dimer association.