* To whom correspondence should be addressed.
Received October 1, 2002
Using site-directed mutagenesis and steady-state kinetic measurements, the functional role of the conserved glycine 127 in a human vaccinia H1-related phosphatase (VHR) was investigated. The mutations of Gly127 to Ala and Pro resulted in a significant decrease in kcat/Km, and increase in Ki for arsenate, indicating that flexibility at the Gly127 site has a large effect on substrate binding and catalytic activity. No substantial decrease in kcat/Km and increase in Ki values were observed for G127 deletion mutant. This showed the conformational flexibility of the PTP loop also affected the enzymatic activity of VHR. Our data suggest that the flexibility of the PTP loop in VHR is probably controlled by Gly127, and that even subtle changes in the loop flexibility may interfere with substrate binding and enzymatic reaction.
KEY WORDS: VHR, site-directed mutagenesis, steady-state kinetics, PTP loop, flexibility