Engineering Specificity for Folate into Dihydrofolate Reductase from Escherichia coli

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• 作者：Bruce A. Posner ; Luyuan Li ; Richard Bethell ; Tomoko Tsuji ; and Stephen J. Benkovic
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：February 6, 1996
• 年：1996
• 卷：35
• 期：5
• 页码：1653 - 1663
• 全文大小：784K
• 年卷期：v.35,no.5(February 6, 1996)
• ISSN：1520-4995

文摘

Despite several similarities in structure and kinetic behavior,the bacterial and vertebrate formsof the enzyme dihydrofolate reductase (DHFR) exhibit differentialspecificity for folate. In particular,avian DHFR is 400 times more specific for folate than theEscherichia coli reductase. We proposed toenhance the specificity of the E. coli reductasefor folate by incorporating discrete elements ofvertebratesecondary structure. Two vertebrate loop mutants, VLI and VLIIcontaining 3-7 additional amino acidinsertions, were constructed and characterized by using steady-statekinetics, spectrofluorimetricdetermination of ligand equilibrium dissociation constants, andcircular dichroism spectroscopy. Remarkably, the VLI and VLII mutants are kinetically similar to wild-typeE. coli reductase when dihydrofolateis the substrate, although VLII exhibits prolonged kinetic hysteresis.Moreover, the VLI dihydrofolatereductase is the first mutant form of E. coliDHFR to display enhanced specificity for folate[(k_cat/K_m)_mutant/(k_cat/K_m)_wt= 13]. A glycine-alanine loop (GAL) mutant was alsoconstructed to test the design principlesfor the VLI mutant. In this mutant of the VLI reductase, all ofthe residues from positions 50 to 60,except the strictly conserved amino acids Leu-57 and Arg-60, wereconverted to either glycine or alanine.A detailed kinetic comparison of the GAL and wild-type reductasesrevealed that the mutations weakenthe binding by both cofactor and substrate by up to 20-fold, but undersaturating conditions the enzymeexhibits a k_cat value nearly identical to thatof the wild type. The rate of hydride transfer is reducedbya factor of 30, with a compensating increase in the dissociation ratefor tetrahydrofolate. Although keystabilizing interactions have been sacrificed (it shows no activitytoward folate), the maintenance of thecorrect register between key residues preserves the activity of theenzyme toward its natural substrate.Collectively, neither specific proximal point site mutations norlarger, more distal secondary structuralsubstitutions are sufficient to confer a specificity for folatereduction that matches that observed with theavian enzyme. This is consistent with the hypothesis that theentire protein structure must contributeextensively to the enzyme's specificity.