Abstract
The reaction of recombinant chlorophyll synthase from Avena sativa, expressed in Escherichia coli, was investigated. To verify the identity of the recombinant and native enzymes, reaction rates were determined for both enzyme preparations with several chlorophyllide analogs. The rates of esterification of these modified substrates ranged from 0 to 100% of the rate with the natural substrate, and were nearly identical for both enzyme preparations. The Lineweaver Burk plot for variation of both chlorophyllide a and phytyl diphosphate concentration showed parallel lines, indicative of a 'pingpong' mechanism. Preincubation with phytyl diphosphate exhibited an initial rapid reaction phase, which did not occur after preincubation with chlorophyllide. We conclude that the tetraprenyl diphosphate must bind to the enzyme as the first substrate and esterification occurs when this preloaded enzyme meets the second substrate, chlorophyllide. Approximately 10 17% of the recombinant enzyme were preloaded with phytyl diphosphate under the experimental conditions. The rapid reaction phase is also observed for the chlorophyll synthase reaction in etiolated barley leaves in addition to the wellknown slow phase. This indicates that preloading of the enzyme with tetraprenyl diphosphate is also the basis for the reaction in vivo.
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