Pre-Loading of Chlorophyll Synthase with Tetraprenyl Diphosphate Is an Obligatory Step in Chlorophyll Biosynthesis
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H. C. Schmid
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.
Copyright © 2002 by Walter de Gruyter GmbH & Co. KG
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- Mechanism of Inhibition of RNA Polymerase I Transcription by DNA-dependent Protein Kinase
- Evidence for Haploinsufficiency of the Human HNF1α Gene Revealed by Functional Characterization of MODY3-Associated Mutations
- In vitro and in vivo Stability of the 2ζ2 Protein Complex of the Broad Host-Range Streptococcus pyogenes pSM19035 Addiction System
- Effects of Antineoplastic Agents on Cytoplasmic and Membrane-Bound Heat Shock Protein 70 (Hsp70) Levels
- Characterization of the VPS10 Domain of SorLA/LR11 as Binding Site for the Neuropeptide HA
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