Our overall thrust is to study the linkage between biological structure and function, using the broad array
of chemical, physical, and biological tools that are now available. Our major efforts fall in four principal areas.
Yeast and E. coli inorganic pyrophosphatases (PPases)
Our previous work on native enzyme has involved determination of an overall kinetic scheme, evaluation of microscopic
rate constants, identification of active site residues through chemical modification, and determination of metal-ion:phosphoryl
ligand and metal-ion:metal ion distances at the enzyme active site through NMR and EPR measurements. Now, in conjunction
with ongoing X-ray crystallographic studies, we are determining the effects of mutation amino acid residues at
the active sites of both enzymes on specific aspects of enzyme function and structure.
E. coli ribosomes
We make extensive use of photoaffinity labeling to localize functional sites on this highly complex enzyme. With
this approach we have determined the identities of ribosomal proteins and RNA bases within ribosomal RNA (rRNA)
at binding sites for several ribosomal antibiotics (e.g., puromycin, tetracycline, chloramphenicol), for tRNA for
initiation factor IF-3, and for oligoDNAs complementary to single-stranded regions of rRNA. We also use reconstitution
methods to prepare ribosomal subunits that have reporter functions specifically incorporated at given sites within
the ribosome. A particular example of such reporter groups are haptens. Electron microscopic examinations of the
complex between the hapten and its corresponding antibody molecule allows three- dimensional localization of the
site of hapten incorporation into the ribosome.
Serine proteinase inhibitors ("serpins")
Serpins are known to be of great importance for inflammation process in mammals. We seek to understand the structural
basis for the specificity of interaction of these serpins with a variety of serine proteases, using a combination
of chemical modification and genetic engineering approaches to both alter and enhance such specificity. This work
involves a very active collaboration with faculty in Penn's School of Medicine who are testing clinical applications
of serpins and point-specific serpin mutants.
Ribonucleotide reductase (RR)
RR catalyzes the reduction of nucleoside diphosphates to deoxynucleoside diphosphates and is the key enzyme controlling
the rate of DNA synthesis. As such it is highly regulated and is a target enzyme for inhibitors of viral and parasitic
diseases. Our studies focus on the RRs derived from mammalian cells and from plasmodium, the malarial parasite.
We are investigating subunit:subunit interaction within this enzyme, genetic engineering and chemical modification
approaches to the study of structure and function, and the development of novel and specific enzyme inhibitors.
Three-dimensional structure of the active site of E. coli inorganic pyrophosphatase.
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