Pre-steady-state kinetic analysis of recombinant arabidopsis NADP : Nitrate reductase - Rate-limiting processes in catalysis

Recombinant Arabidopsis NADH:nitrate reductase was expressed in Pichia past oris using fermentation. Large enzyme quantities were purified for pre-stea dy-state kinetic analysis, which had not been done before with any eukaryot ic nitrate reductase, Basic biochemical properties of recombinant nitrate r eductase were similar to natural enzyme forms. Molybdenum content was lower than expected, which was compensated for by activity calculation on molybd enum basis. Stopped-flow rapid-scan spectrophotometry showed that the enzym e FAD and heme were rapidly reduced by NADH with and without nitrate presen t. NADPH reduced FAD at less than one-tenth of NADH rate. Reaction of NADH- reduced enzyme with nitrate yielded rapid initial oxidation of heme with sl ower oxidation of flavin, Rapid-reaction freeze-quench EPR spectra revealed molybdenum was maintained in a partially reduced state during turnover. Ra pid-reaction chemical quench for quantifying nitrite production showed that the rate of nitrate reduction was initially greater than the steady-state rate, but rapidly decreased to near steady-state turnover rate. However, ra tes of internal electron transfer and nitrate reduction were similar in mag nitude with no one step in the catalytic process appearing to be much slowe r than the others. This leads to the conclusion that the catalytic rate is determined by a combination of rates with no overall rate-limiting individu al process.