CAREY LAB

PROJECTS AND COLLABORATIONS

Some of the key research projects and collaborations are listed below.

Collaborators: D. Samols , Dept. of Biochemistry, CWRU, F. Sonnichsen, Dept. of Physiology and Biophysics, CWRU

The enzyme transcarboxylase (TC), from P. shermanii, transfers a unit of CO2 from methylmalonyl-CoA to pyruvate forming oxalacetate. A key intermediate of this reaction involves the 1.3 S subunit of TC which contains a biotin cofactor attached to Lys-89; it is to this cofactor that CO2 binds during the transfer reaction. The primary purpose of this research is to describe the structure, electron distribution and mechanism of action of the CO2-biotin moiety within the CO2-biotin-1.3 S TC complex. The 1.3 S subunit of TC is a small protein of only 123 residues and emerging technologies in Raman, NMR and FTIR spectroscopies are allowing us to delineate the key structural and mechanistic factors.

SCHEMATIC MODEL OF TRANSCARBOXYLASE

Collaborator: D. Hilvert, The Scripps Research Institute

These semisynthetic enzymes have great potential in areas such as peptide bond synthesis. The Raman research is exploring how active site electric fields aid catalysis and how they can be switched on and off by changes in substrate conformation. 

 

Collaborator: B.A. Malcolm, University of Alberta

This is an enzyme from the picornaviridae family; the latter is responsible for a number of human and animal pathologies. The enzyme has structural and chemical properties which are hybrid of the classic serine and cysteine proteases and our interest is on understanding how this mixture of properties define mechanism. 

Collaborator: D. Dunaway-Mariano, University of Maryland

Soil-dwelling bacteria are developing enzyme systems which are able to use pollutants, such as chlorinated hydrocarbons, as substrates. The chemistry undertaken by these enzymes is often difficult and there is much interest in understanding mechanism from both intellectual and practical standpoints. We have been collaborating on mechanistic studies of a dehalogenase from Pseudomonas that removes chlorine from 4-chlorobenzoyl-CoA. 

 

Collaborator: V.E. Anderson, Dept of Biochemistry, CWRU

Crotonase, enoyl-CoA hydratase, catalyses the syn addition of water across a ,b unsaturated CoA thioesters. Interestingly, it has a strong homology to dehalogenase. Research in the Carey lab. has focused on the electron polarizing effects on the active site on bound substrates or substrate analogs. 

Collaborators: B. Palfey (PHBH) University of Michigan, A. Jaffe (PBGS) Fox Chase Cancer Center, Philadelphia

Research on these enzymes is at an early stage but both have shown that it is now possible to obtain Raman difference spectra on enzyme-ligand complexes which hither to have been inaccessible to analysis. Recent technical improvements permit the obtention of high quality Raman data free from interference from fluorescence background. The spectra reveal chemical information on both the bound ligand and the encompassing protein and have high potential for providing detailed and novel mechanistic information.

"> 

 

The Carey lab has a long history of pioneering the use of Raman spectroscopy to probe the structure and dynamics of enzyme-substrate complexes.  A recent development has been to carry out these study in single protein crystals, an approach termed "Raman crystallography."  Thus,  compounds such as drug candidates can be infused into crystals and, by employing Raman difference spectroscopy, Raman data are obtained for the drug bound in the active site.  Analysis of the data yields structural information that often complements that obtained by other biophysical methods.  "Raman crystallography" has two major advantages:  the Raman data are much higher in quality compared to those obtained in aqueous solution and there is a very high degree of synergism with X-ray crystallographic analysis.  Thus, Raman can provide vital information to the crystallographer on chemical events occurring in the crystal that enable him to select the optimum conditions prior to flash freezing and x-ray analysis.

Two characteristics mark the projects within the Carey lab.  Firstly, they are highly interdisciplinary, ranging from protein chemistry to spectroscopy to quantum mechanical calculations.  Secondly, they involve close collaborations with other groups locally and nationally.

 

Transcarboxylase

The enzyme transcarboxylase (TC), from P. shermanii, transfers a unit of CO2 from methylmalonyl-CoA to pyruvate forming oxaloacetate.  Intriguingly, TC is a large multi-subunit complex of 1.2 million Daltons molecular weight.  Extensive NMR studies have been undertaken on the small biotinylated subunit (in collaboration with the Sonnichsen group at Case) and combined Raman and X-ray crystallographic analyses have been performed on substrate complexes of the 12S and 5S subunits (in collaboration with the Yee group at Case).  The ultimate goal is to achieve a detailed molecular-level understanding of the intact nanomachine.


A schematic model of transcarboxylase.

 

Beta-lactamase and Drug Resistance

The reactions between three clinically relevant inhibitors, tazobactam, sulbactam, and clavulanic acid, and SHV beta-lactamase can be followed in single crystals using a Raman microscope. The data are far superior to those obtained for the enzyme in aqueous solution and allow us to identify species on the reaction pathway and to measure the rates of the accumulation and decay of these species. A key intermediate on the reaction pathway is an acyl enzyme formed between Ser70 and the lactam ring's C=O group. By using the E166A deacylation deficient variant of the enzyme, we were able to focus on the process of acyl enzyme formation. The Raman data show that all three inhibitors form an enamine-type acyl enzyme.  The Raman data also demonstrate that the lactam ring opens prior to enamine formation since the lactam ring carbonyl peak disappears prior to the appearance of the enamine band. Tazobactam appears to form approximately twice as much enamine intermediate as sulbactam and clavulanic acid, which correlates with its superior performance in the clinic, a finding that may bear on future drug design.  The Raman data set the conditions that enable our crystallographer colleagues (the van den Akker group at Case) to trap well-defined intermediates for structural analysis.  By comparing normal and drug resistant beta-lactamases, we will elucidate the molecular basis for drug resistance.


SHV-1 betalactmase crystal structure