Rapid Scanning Stopped Flow Spectrophotometer for Enzymology

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Mechanistic and kinetic studies of enzymes involved in the metabolism of aromatic hydrocarbons, degradation of alkylamines, and respiration will be undertaken. The enzymes include the flavocytochrome p-cresol methylhydroxylase (Pseudomonas putida), the quinoprotein methylamine dehydrogenase (methylotrophic bacteria), copper-containing quinoprotein amine oxidases (human and Arthrobacter globoformis), and iron-sulfur flavoproteins succinate dehydrogenase (mammalian and bacteria) and fumarate reductase from Escherichia coli. The numbers, types, and redox potentials of the organic and metal ion cofactors are already defined. The majority of these enzymes have been cloned to allow for alteration of their properties by genetic manipulation. The available x-ray structures of many of the proteins studied will guide some of the research. It is proposed that the requested rapid-scanning stopped-flow spectrophotometer be used to define events occurring on substrate/ligand binding to enzymes, and the properties of the individual cofactors in integrated functions. It is also proposed that the instrument be used as a rapid-recording, dual-wavelength spectrophotometer for essential quinol oxidation reduction assays.