Our overall research goal is to understand the mechanisms used by biological catalysts, both proteins and nucleic acids, to achieve high efficiency and stringent specificity. We are particularly focussed on the mechanism of medically important metalloenzymes. We are investigating the catalytic mechanism and specificity of protein farnesyltransferase and protein geranylgeranyltransferase I. These enzymes catalyze the addition of a prenyl group onto a C-terminal cysteine on a variety of substrates involved in signal transduction. Compounds that inhibit FTase are being investigated as possible antitumor agents. A second enzyme, UDP-3-O-acyl-GlcNAC deacetylase (LpxC) is a zinc metalloenzyme that catalyzes the first committed step in the pathway to form Lipid A, a crucial component of the outer membrane of gram negative bacteria. Inhibitors of this enzyme have antibacterial activity. To further the development of novel inhibitors we are elucidating detailed structure-function relationships in the active site of these proteins using mutagenesis, kinetic analysis, X-ray crystallography, and spectroscopic studies. Additionally, we have begun mechanistic studies of two related enzymes, histone deacetylase and protein palmitoyltransferase. For all of the metalloenzymes, a key question is the identify of the in vivo metal ion and whether metal switching is an important regulatory mechanism. Finally, we are developing methods for high-throughput screens of protein-bound transition metal ions for use in assaying all of the proteins in the yeast proteome to identify the yeast "metallome".
We also investigating the catalytic modes of ribozymes compared to proteins by determining the structure and mechanism of ribonuclease P (RNase P), a ribonucleoprotein complex that catalyzes the cleavage of tRNA precursors. We have demonstrated that the protein component enhances the catalytic efficiency by interacting with both P RNA and pre-tRNA. In the future, we will elucidate the structure of the holoenzyme using fluorescence resonance energy transfer, crystallography and spectroscopy. Finally, we will investigate the mechanism of yeast RNase P (in collaboration with Dr. D. Engelke) which contains one RNA and multiple protein subunits and purify, clone and characterize the RNase P from mammalian mitochondria which is proposed to be a protein catalyst.
Zinc, iron and copper ions are proposed to play important biological roles, especially in neurobiology, as well as playing important roles in the development of a number of diseases, including diabetes. Furthermore, a number of metals, such as lead and cadmium, are toxic. We are investigating the mechanisms of metal homeostasis and metal toxicity using a combination of biochemistry, genetics and imaging. To this end, we are redesigning the zinc metalloenzyme, carbonic anhydrase II, to optimize a fluorescent biosensor for measuring and imaging "readily exchangeable" metal ions in complex biological mixtures, such as cells, plasma and sea water and are developing similar sensors to measure cellular iron concentrations. Additionally, we are using X-ray fluorescence microprobe imaging to image total metal ions in wild-type and mutant yeast cells. These imaging methods are being used to understand basic mechanisms of metal homeostasis. Finally, we are examining the metal content and the mechanisms of metal insertion into proteins in vivo using biochemistry and analysis of libraries of deletion mutants. These studies should lead to a better understanding of the functions and regulation of biological transition metals.
B. A. – Chemistry, 1978, Carleton College
Ph. D. – Biochemistry, 1984, Brandeis University
Postdocoral – 1984-1987, Pennsylvania State University