David H.T. Harrison, PhD, Professor, was appointed January 10, 2010 to the COP. Dr. Harrison took his Bachelor’s and Master’s degrees in Chemistry. He completed an MPhil and PhD in Molecular Biochemistry and Biophysics at Yale University. During a brief hiatus from Yale (1986-87), Dr. Harrison was a Research Scientist in Protein Engineering at Eastman Kodak Company. Later, he was the Juvenile Diabetes Postdoctoral Fellow in the laboratory of Dr. Gregory Petsko at Brandeis University. His first academic position was at the Medical College of Wisconsin. In 2003, he moved to RFUMS to take an Associate Professor position in the Department of Biochemistry and Molecular Biology. He also became the Director of the X-Ray core facility in the Rosalind Franklin Structural Biology Laboratory. His research career has been productive, with grant funding from NIH, NSF and private foundations. He has published in international, peer-reviewed journals and is a member of several national professional societies, including the AACP. He has extensive experience in the teaching of Biochemistry and Molecular Biology to professional students and has mentored several of these students in his laboratory. He serves the university on many committees and is active in community outreach.

Curriculum Vitae

Courses

• Pharmaceutics I: Introduction to Pharmaceutical Sciences
• Biochemical Principles for Pharmacy
• Life-Long Learning Seminar

Research

Electron density for two Alrestatin molecules bound to the active site of human aldose reductase

Research in my laboratory is primarily concerned with molecular recognition, or answering the question of "how does molecule A recognize and bind molecule B?" Our research focuses on proteins that are involved in diabetes and other debilitating diseases. We use the methods of mechanistic enzymology, small molecules organic synthesis, site-directed mutagenesis, and X-ray diffraction to establish on an atomic level how each protein functions.

Our most developed area of research is on the diabetes related enzyme, human aldose reductase. As a postdoctoral fellow I determined the structure and deduced the mechanism of this enzyme. We are now engaged in mapping a region known as the "specificity" determinant for use in the design of highly specific small molecule inhibitors. The specificity determinant was found when we determined the structure of aldose reductase with the drug Alrestatin bound to the active site. This structure is remarkable in that there are two drug molecules bound in the same active site cavity. The carboxylate of one molecule is positioned in the manner that was expected from the previously determined mechanism of inhibition. The carboxylate of the other drug molecule is binding to a region of the enzyme that is unique to aldose reductase, and not found in other members of the aldo-keto reductase super-family.

The stacking arrangement of the Alrestatin molecules is reminiscent of the structure of ferrocene acetic acid derivatives. Using a combinatorial chemistry approach, we will attach a ferrocene acetic acid to a random peptides and select the peptides which bind aldose reductase tightly, and that do not bind the related enzyme aldehyde reductase. The structure of aldose reductase bound to the appropriate ferrocene acetic acid peptide derivative will be determined, and the geometries between the functional groups will be used in designing highly specific inhibitors.

The specificity determinant and other sites will also be probed using the new technique of "solvent mapping." The diffraction pattern of an organic solvent soaked cross-linked protein crystal is measured. The location of the bound solvent molecules are mapped to the surface of the protein. After a number of solvents are mapped to the surface of the protein, a unique three-dimensional pattern of functional group binding sites emerge. This information is used to design highly specific inhibitors. This technique is a valuable teaching aid, as it allows a student to experience many of the facets of protein crystallography (crystal growth and manipulation, data collection, and electron density fitting) during a three month period of time.

A second project in my laboratory focuses on methylglyoxal synthase an enzyme found on an alternate branch of the glycolytic pathway. Methylglyoxal syntase appears to be fundamental to bacterial life, yet not present in eukaryotes. The enzyme catalyzes the irreversible conversion of dihydroxy acetone phosphate (DHAP) to methylglyoxal and inorganic phosphate. This project is typical of future studies in my laboratory as we have purified the endogenous protein, used its N-terminal sequence to clone and sequence the gene, used site-directed mutagenesis to identify residues critical for catalysis, and we have obtained crystals and are in the process of determining the three dimensional structure. Surprisingly, the enzyme appears to be unrelated to triose phosphate isomerase, an enzyme that converts DHAP to glyceraldehyde phosphate. It will be exciting to learn how these two enzymes which bind the same substrate control their reaction coordinate to avoid making the other enzyme's product. We have also crystallized the next enzyme on this alternate pathway, the ubiquitous enzyme, glyoxalase I, which is important since it detoxifies a major class of cancer chemotherapeutic agents. Our goal is to help in the development of novel glyoxalase I inhibitors that will allow these chemotherapeutic agents to function.

In collaboration with Dr. Henry Miziorko, at MCW, I am also working on the structure of the bacterial form of the photosynthetic protein phosphoribulokinase (PRK). While this enzyme is essential for life on the planet and controls the rate determining step in CO2 uptake, this project also addresses some of the broader issues of phosphoryl transfer common to all kinases. Having successfully determined the structure of the apo-enzyme, we are currently trying to obtain crystals with substrates, products, allosteric effectors, or inhibitors bound, and in this way create some of the "still frames" in a three dimensional movie of the enzyme at work. In collaboration with Dr. Richard Sabina, also at MCW, we have successfully crystallized the AMP-deaminase. This protein, important in muscular disorders, is functionally similar to adenosine daminase, yet the enzymes share little sequence similarity and does not catalyze the deamination of the other's substrate.

Publications

1. Aluvila S, Sun J, Harrison DH, Walters DE, Kaplan RS (2010) “Inhibitors of the mitochondrial citrate transport protein: validation of the role of substrate binding residues and discovery of the first purely competitive inhibitor” Mol Pharmacol. 77:26-34
2. Remani S, Sun J, Kotaria R, Mayor JA, Brownlee JM, Harrison DH, Walters DE, Kaplan RS (2008) “The yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate” J Bioenerg Biomembr. 40: 577-585
3. June Brownlee, Panqing He, Graham R. Moran, and David H. T. Harrison (2008) “Two Roads Diverged: The Structure of Hydroxymandelate Synthase from Amycolatopsis orientalis in Complex with 4-Hydroxymandelate” Biochemistry 47: 2002-2013
4. Brownlee, J.M., Calrson, E., Milne, A.C., Pape, E., and Harrison, D. H. T. (2006) “Structural and thermodynamic studies of simple aldose reductase-inhibitor complexes” Bioorganic Chemistry 34:424-444.
5. Bohren, K. M., Messmore, J., Milne, A., Gabbay, K. H., and Harrison, D. H. T. (2005) “Hinges and Latches: The Structure of Human Apo Aldose Reductase and Its Relationship to the Enzyme Mechanism” Biochemica & Biophysica Acta 1748:201-212.
6. Park, M., Lin, L., Thomas, S., Braymer, H. D., Smith, P. M., Harrison, D. H., and York, D. A. (2004) “The F1-ATPase beta-subunit is the putative enterostatin receptor” Peptides. 25(12):2127-33.
7. Theisen M. J., Misra I., Saadat D., Campobasso N., Miziorko H. M. and Harrison D. H. T. (2004) “3-hydroxy-3-methylglutaryl-CoA synthase intermediate complex observed in ‘real-time’” Proceedings of the National Academe of Sciences 101:16442-16447
8. Brownlee, J. M., Johnson-Winters, K., Harrison, D. H. T., and Moran, G. R. (2004) “Structure of the Ferrous Form of (4-Hydroxyphenyl)pyruvate Dioxygenase from Streptomyces avermitilis in Complex with the Therapeutic Herbicide, NTBC” Biochemistry (Accelerated Publication) 43(21):6370-6377.
9. Marks, G. T., Susler, M., and Harrison D. H. T. (2004) “Mutagenic Studies on Histidine 98 of Methylglyoxal Synthase: Effects on Mechanism and Conformational Change” Biochemistry 43(13):3802-3813.
10. Zhang, X., Harrison D. H. T., Cui Q. (2002) “The functional specificities of methylglyoxal synthase (MGS) and triosephosphate isomerase (TIM) are not due to stereoelectronic effects: A combined QM/MM analysis” Journal of the American Chemical Society 124(50):14871-14878
11. Marks, G., Harris, T. K., Massiah, M. A., Mildvan, A. S., and Harrison, D. H. T. (2001) “Mechanistic Implications of Methylglyoxal Synthase Complexed with Phosphoglycolohydroxamic Acid as Observed by X-Ray Crystallography and NMR Spectroscopy” Biochemistry 40: 6805-6818
12. Saadat, D. and Harrison D. H. T. (2000) “Mirroring Perfection: The Structure of Methylglyoxal Synthase Complexed With the Competitive Inhibitor 2-Phosphoglyocolate.” Biochemistry 39: 2950-2960.
13. Kung, G., Runquist, J. A., Miziorko H. M., and Harrison, D. H. T. (1999) “The Identification of the Allosteric Regulatory Site in Bacterial Phosphoribulokinase” Biochemistry 38:15157-15165.
14. Runquist, J. A., Harrison, D. H. T., Miziorko H. M. (1999) “R. Sphaeroides Phosphoribulokinase: Identification of Lysine-165 as a Catalytic Residue and Evaluation of the Contributions of Invariant Basic Amino Acids to Ribulose 5-Phosphate Binding” Biochemistry 38:13999-14005.
15. Saadat, D. and Harrison D. H. T. (1999) “The Crystal Structure of Methylglyoxal Synthase from Escherichia coli” Structure with Folding & Design 7:309-317.