Functional role of a mobile loop of Escherichia coli dihydrofolate reductase in transition-state stabilization.
The function of a highly mobile loop in Escherichia coli dihydrofolate reductase was studied by constructing a mutant (DL1) using cassette mutagenesis that had four residues deleted in the middle section of the loop (Met16-Ala19) and a glycine inserted to seal the gap. This part of the loop involves residues 16-20 and is disordered in the X-ray crystal structures of the apoprotein and the NADP+ binary complex but forms a hairpin turn that folds over the nicotinamide moiety of NADP+ and the pteridine moiety of folate in the ternary complex [Bystroff, C., & Kraut, J. (1991) Biochemistry 30, 2227-2239]. The steady-state and pre-steady-state kinetics and two-dimensional 1H NMR spectra were analyzed and compared to the wild-type protein. The kinetics on the DL1 mutant enzyme show that the KM value for NADPH (5.3 microM), the KM for dihydrofolate (2 microM), the rate constant for the release of the product tetrahydrofolate (10.3 s-1), and the intrinsic pKa value (6.2) are similar to those exhibited by the wild-type enzyme. However, the hydride-transfer rate declines markedly from the wild-type value of 950 s-1 to 1.7 s-1 for the DL1 mutant and when taken with data for substrate binding indicates that the loop contributes to substrate flux by a factor of 3.5 x 10(4). Thus, the mobility of loop I may provide a mechanism of recruiting hydrophobic residues which can properly align the nicotinamide and pteridine rings for the hydride-transfer process (a form of transition-state stabilization).(ABSTRACT TRUNCATED AT 250 WORDS)
Li
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Department of Chemistry, Pennsylvania State University, University Park 16802.
Falzone
C J
CJ
Wright
P E
PE
Benkovic
S J
SJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016254:Mutagenesis, Insertional; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism
D013763:Tetrahydrofolates; C030371:5,6,7,8-tetrahydrofolic acid; D009249:NADP; D003903:Deuterium; D013762:Tetrahydrofolate Dehydrogenase