Inactivation of the human cytomegalovirus protease by diisopropylfluorophosphate

Abstract

Publisher Summary Cytomegalovirus (CMV) protease is a serine protease, and labeling with diisopropylfluorophosphate (DFP) has identified Ser132 as the active site serine. The structure of the CMV protease containing the diisopropylphosphorylserine at residue 132 (DIP-CMV protease) is likely to resemble that of the tetrahedral transition-state intermediate. As the structure of the DFP-treated serine proteases resembles that of the tetrahedral transition-state intermediate, and the inactivated enzyme would not be susceptible to autoproteolysis, production of DIP-CMV protease would be useful for structure based drug design. The concentrations of DFP sufficient to yield stoichiometric incorporation of inhibitor at the active site of CMV protease, also resulted in substantial incorporation of DIP at a second site or sites. This heterogeneous incorporation would preclude crystallographic studies. For this reason, this chapter has attempted to optimize the conditions for inactivation of CMV protease with DFP, to produce pure DIP-CMV protease with minimum second site incorporation. Initial studies indicated that there was no loss of protease activity in the control samples, even when incubated for 23 hours at 22°C. A 3.8 hour incubation was sufficient to completely inactivate 180 μM protease in the presence of 4.3 mM DFP, while a 2.4 fold excess of the inhibitor inactivated only 50% of the enzyme. This indicates that CMV protease is less reactive with DFP than trypsin or chymotrypsin, and requires an order of magnitude excess of the organophosphate to achieve complete inactivation.