Most bacteria, yeast, fungi, algae and higher plants can use inorganic sulfate as their sole source of sulfur for the biosynthesis of cysteine, methionine, and a variety of organic sulfur-containing coenzymes. The objective of this research is to establish the structure-function relationships of the enzymes which catalyze the early steps of sulfate assimilation. The enzymes ATP Sulfurylase, APS kinase, and PAPS reductase will be studied. The first two catalyze the two-step ATP- dependent "activation" of S042- forming 3'- phosphoadenosine- 5' -phosphosulfate (PAPS). The last named enzyme (actually a multicomponent system) catalyzes the NADPH- and thioredoxin- dependent reduction of PAPS to inorganic sulfite. Specific aims include (a) determining the kinetic and chemical mechanisms, (b) identifying aminoacyl residues at the active site, and (c) exploring the potential interaction of the two sulfate activating enzymes to form a "PAPS synthetase" complex. The experiments will be conducted with enzymes purified from the filamentous fungus Penicillium chrysogenum and from spinach leaf. Parallel studies will be performed on the enzymes from P. duponti (a thermophile) with the objective of identifying the structural features responsible for the remarkable heat stability of thermophile enzymes. Enzyme kinetics, protein chemistry, and molecular biology methods will be used. Inorganic sulfate is reduced to a state suitable for incorporation into sulfur containing amino acids and other factors by a complex pathway in plants and microorganizms. The investigator employs a sophisticated mathematical approach to the study of these enzymes. He wishes to analyze the chemical mechanisms by which they carry out the reactions involved.
