The research plan proposes the study of the structure and function of metalloenzymes in three specific areas. 1) Continuation of a detailed correlation of crystal and solution structure for alkaline phosphatase of E. coli. This requires the preparation and crystal structure determination of homogeneous forms of the 2 phosphoenzyme intermediates. NMR and crystal structure of two mutant forms, Ser 102-Cys 102 and Ser 102-Ala 102 will be undertaken. Using a cloned gene for E. coli alkaline phosphatase, conditions have been found under which large quantities of preenzyme containing the signal peptide are formed by phosphate starvation. The preenzyme has been isolated in large quantities by gentle detergent extraction from the membrane. The quaternary and tertiary structure, metal binding and phosphate binding properties of the preenzyme are being determined as a prototype for structural differences between preenzymes and processed enzymes or proteins. This process has important implications for a variety of health-related cellular processes, i.e. antibody secretion, digestive enzyme transport and the regulation of hormone release. The crystallization and crystal structure of the alkaline phosphatase preenzyme will be attempted. 2) 113Cd NMR as a probe of metal binding sites for Zn(II) Ca(II) and Mg(II) sites in metalloproteins by substituting 113Cd(II) in these sites. 113Cd NMR as a probe of the allosteric T to R transition in aspartate transcarbamylase will be explored. An understanding of the catalytic mechanisms of enzymes, many of which contain an essential metal ion, is essential to the understanding of metabolic control. 3) A comparative study of Zn(II) and non-Zn(II) RNA polymerases. Studies in this laboratory have shown that the simple monomeric T7 RNA polymerase does not require Zn(II) for activity, while the multisubunit a2 beta beta' E. coli RNA polymerase has been confirmed to contain 2.0 plus or minus 0.1 gm at Zn/mole, non-exchangeable under most conditions. The structural, allosteric and possible catalytic role of Zn(II) in the multisubunit polymerases is being investigated by spectroscopic techniques and by cloning and overproducing the separate subunits. The understanding of the structure and allosteric control features of RNA polymerases are essential to the understanding of the control of gene expression, a process important in controlling cell growth and development.
