Pre-loading of chlorophyll synthase with tetraprenyl diphosphate is an obligatory step in chlorophyll biosynthesis
Autor: | Siegrid Schoch, Heidi C. Schmid, Wolfhart Rüdiger, Ulrike Oster, Valentina Rassadina |
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Rok vydání: | 2003 |
Předmět: |
Chlorophyll
Avena Stereochemistry Clinical Biochemistry Blotting Western medicine.disease_cause Biochemistry Maltose-Binding Proteins law.invention Reaction rate chemistry.chemical_compound law medicine Escherichia coli Cloning Molecular Molecular Biology Carbon-Oxygen Ligases Chromatography High Pressure Liquid chemistry.chemical_classification ATP synthase biology Substrate (chemistry) Recombinant Proteins Kinetics Enzyme chemistry Etiolation biology.protein Recombinant DNA Mutagenesis Site-Directed Diterpenes Carrier Proteins |
Zdroj: | Biological chemistry. 383(11) |
ISSN: | 1431-6730 |
Popis: | 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 'ping-pong' mechanism. Pre-incubation with phytyl diphosphate exhibited an initial rapid reaction phase, which did not occur after pre-incubation with chlorophyllide. We conclude that the tetraprenyl diphosphate must bind to the enzyme as the first substrate and esterification occurs when this pre-loaded enzyme meets the second substrate, chlorophyllide. Approximately 10-17% of the recombinant enzyme were pre-loaded 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 well-known slow phase. This indicates that pre-loading of the enzyme with tetraprenyl diphosphate is also the basis for the reaction in vivo. |
Databáze: | OpenAIRE |
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