Enzymes: Catalytic Function, Structure, Engineering, Evolution and Sexual Dimorphism

Enzymes are the primary subject of Dr. Walz's research which involves a variety of cross-disciplinary studies on the structure, function, genetic diversity and regulation of these interesting protein biocatalysts. Projects have ranged from physical-chemical studies on stable, low molecular mass ribonucleases to proteomics of sexual dimorphism.

Current work on ribonuclease T1 (RNase T1; a well characterized extracellular fungal enzyme) includes random combinatorial mutagenesis of functional sites to probe whether local perfection has been achieved by evolution. Initial studies involved the guanine recognition site which directs specific cleavage of RNA. This work revealed mutant forms of the enzyme having dramatic increases and decreases in specificity and catalytic rate. One active mutant was structurally labile which suggested a novel limitation on the evolutionary sequence-space. Studies are underway that are probing the effect of native disulfide bonds on catalysis. This approach is also planned to investigate whether alternate arrangements of multiple amino acid residues at the active site can be catalytically effective on the same structural scaffold. In addition, the complete array of subsites (that influence the catalytic rate) will be studied using oligonucleotide substrates with fluorescently quenched terminal fluorophores. Continuing collaborative work involves the determination of X‑ray crystallographic structures for this enzyme (and mutant versions) complexed with a variety of ligands to probe structure/function relationships. Future research is also planned to explore the biological purpose of this extracellular enzyme using engineered fungal strains not having the RNase T1 gene.

Previous work perfected the use of high resolution two‑dimensional gel electrophoresis to resolve integral membrane proteins. Analysis of liver microsomal membrane subfractions led to the discovery of novel Cytochrome P450 allozymes and closely related isozymes. The metabolism of progesterone (steroid hormone) and warfarin (anti‑clotting drug) by liver microsomes revealed the presence and metabolic importance of novel enzymes which catalyzed the reduction of carbonyl groups by NADPH. These and other xenobiotic metabolizing enzymes in the liver are expressed only in adult, male rats. Two‑dimensional electrophoresis has been used in proteomic studies to test the extent of sexual dimorphism in the liver and other tissues of the rat.

Selected Publications

Kitareewan, S. and Walz, F. G., Jr. (1994) Genetic and Developmental Diversity of Hepatic Cytochromes P450: Warfarin and Progesterone Metabolism by Hepatic Microsomes from Four Inbred Strains of Rat, Drug Metabol. Disp. 22, 607-615.

Apanovitch, D. and Walz, F. G., Jr. (1996) S-Warfarin (11S-OH) and Progesterone (20β-OH) Keto‑Reductases in Rat Hepatic Microsomes are not Identical, Biochim. Biophys. Acta1291, 16-26.

Arni, R. K., Watanabe, L., Ward, R. J., Kreitman, R. J., Kumar, K. and Walz, Jr., F. G. (1999) Three-Dimensional Structure of Ribonuclease T1 Complexed with an Isosteric Analogue of GpU: Alternate Substrate Binding Modes and Catalysis, Biochemistry38, 2452-2461.

Kumar, K. and Walz, Jr., (2001) Probing Functional Perfection in Substructures of Ribonuclease T1: Double Random Mutagenesis Involving Asn43, Asn44, and Glu46 in the Guanine Binding Loop , Biochemistry40, 3748‑3757.

Chitester, B. J. and Walz, Jr., F. G. (2002) Kinetic Studies of Guanine Recognition and a Phosphate Group Subsite on Ribonuclease T 1 Using Substitution Mutants at Glu46 and Lys41. Arch. Biochem. Biophys. 406, 73-77.

Last Updated: 15 June 2006