High expression and steady-state kinetic characterization of methionine site-directed mutants of Escherichia coli methionyl- and selenomethionyl-dihydrofolate reductase.
A high expression system that produces Escherichia coli dihydrofolate reductase (DHFR) at 30% total cellular protein was constructed. This expression vector, named pCOCK, allowed for the purification of nearly 100 mg of homogeneous DHFR from a 11 bacterial culture. A simple, single Q-Sepharose anion exchange column purification was developed on an FPLC instrument. Methionine site-directed mutants were constructed in DHFR to assess the role of Met within the enzymes. These mutants consisted of a Met16leucine (Leu), Met20Leu, Met42Leu, Met92Leu, Met16,20Leu and Met16,20,42Leu. Steady-state kinetic studies showed that the Met16Leu, Met42Leu and Met92Leu mutants possessed essentially the same kcat, Km(DHF) and Km(NADPH) as that of wild-type (wt) DHFR (13.7 s-1, 0.97 microM and 2.52 microM, respectively). Mutants which contained a Leu at position 20 possessed substantially elevated specific activity and kcat values. The specific activity and kcat of wt, Met20Leu, Met16,20Leu and Met16,20,42Leu were 45.9, 92.7, 90.2 and 172 mumol/min/mg and 13.7, 24.6, 25.2 and 52.7 s-1, respectively. Upon substitution of Met by selenomethionine (SeMet) in the aforementioned mutants, further information as to the effect of SeMet incorporation into proteins was ascertained. Steady-state kinetic parameters of the SeMet substituted Met16Leu, Met20Leu, Met42Leu and Met92Leu mutants were nearly identical to those of their Met containing counterparts. These data indicate that Met apparently has a limited role in the protein structure and function of DHFR and that SeMet incorporation has no effect on the steady-state kinetic constants of DHFR.
Shaw
D
D
Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208, USA.
Odom
J D
JD
Dunlap
R B
RB
D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D008715:Methionine / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D012645:Selenomethionine / Q000737:chemistry*; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics