Harvey F. Fisher, Ph.D., Professor

Department of Biochemistry and Molecular Biology

hfisher@kumc.edu
Phone: (816) 861-4700, ext. 5-7156
FAX: (816) 861-1110
Office location: Kansas City VA Medical Center

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Education and Experience

University of Chicago, Chicago, Illinois, Ph.D., 1952; Columbia University (Postdoctoral Fellow); University of Wisconsin (Project Associate); University of Massachusetts (Instructor); E.B. Ford Institute of Medical Research (Senior Research Associate); Director, Laboratory of Molecular Biochemistry, VA Medical Center

 

Major Research Interests

The Laboratory of Molecular Biochemistry is devoted to the study of enzymatic catalysis. Our work pursues three lines of inquiry:

• the determination of the chemical mechanism of the glutamate dehydrogenase reaction
• the energetics of this reaction; and
• the relationship of the observed energetics to the resolved mechanistic features.
  
  

Our mechanistic component relies heavily on following the time courses of optically identified and resolved intermediate complexes using newly developed transient-state approaches (including the detection of proton release steps and the use of time and signal dependent transient-state kinetic isotope effects. The energetic component of our work involves a variety of calorimetric approaches. The third component aims at the creation of a viable theory capable of integrating kinetic, chemical, and thermodynamic observations into a unified picture of the operation of a biological molecular machine.

 

 

Representative Publications

H.F. Fisher. Transient-State Kinetic Approach to Mechanisms of Enzymatic Catalysis, Accounts of Chemical Research, (In Press, 2005).

H.F. Fisher.  Glutamate Dehydrogenase as a Molecular Machine, Advances in Enzymology, (In Press, 2005).

Maniscalco, S.J., J.F. Tally, and H.F. Fisher. The interpretation of multiple-step transient-state kinetic isotope effects.  Arch. Biochem. Biophys. 425.2 (2004): 165-72.

Steven J. Maniscalco, Jon F. Tally, S. Welsh Harris and Harvey F. Fisher. The Direct Measurement of Thermodynamic Parameters of Reactive Transient Intermediates of the L-Glutamate Dehydrogenase Reaction. Journal of Biological Chemistry. 278.18 (2003): 16129-34.

Swapan K. Saha, Jon F. Tally, Steven J. Maniscalco and Harvey F. Fisher. Structure-Function Relationships in the Bovine Glutamate Dehydrogenase Catalyzed Reaction Determined by the Use of a Product Inhibitor in a Resolved Component Transient State Time Course. Submitted to Biochimica et Biophysica Acta (February 2003).

J. F Tally, S. J Maniscalco, H. F. Fisher. Detection of Multiple Active Site Domain Motions in Transient-State Component Time Courses of the Clostridium symbiosum L-Glutamate Dehydrogenase-Catalyzed Oxidative Deamination Reaction. Biochemistry, 41:11284-11293 (2002).

H.F. Fisher and Steven Maniscalco. A Close-Packed Planar 4-Atom Motif Serves as a Variable-Pathway Mechanistic Switching Device in Enzymatic Catalysis, Bioorganic Chemistry, 30:199-209 (2002).

H.F. Fisher, Jon Tally and Steven J. Maniscalco. Stabilization of Non-Covalent Intermediates In Enzymatically Catalyzed Reactions, Biochemical and Biophysical Research Communications 287:343-347 (2001).

H.F. Fisher, Transient State Enzyme Kinetics. (Accounts of Chemical Research, Invited Paper, In Press)

H.F. Fisher. Glutamate Dehydrogenase as a Molecular Machine, Advances in Enzymology, (In Press, 2000).

H.F. Fisher. Protein Ligand Interactions Molecular Basis, Encyclopedia of Life Sciences, Vol. 15, 2000, pp. 496-505. London: Nature Publ. Group. http//www.els.net, June 2000.

S.K. Saha, and H.F. Fisher. The Location of Active Site Opening and Closing Events in the Prehydride Transfer Phase of the Oxidative Deamination Reaction Catalyzed by Bovine Liver Glutamate Dehydrogenase Using a Novel pH Jump Approach. BBA 1431, 261-265 (1999).

H.F. Fisher. Transient State Kinetic Approaches to the Resolution of Enzyme Mechanisms, in Enzymatic Mechanisms, eds. P.A. Frey and D.B. Northup IOS Press 264-277 (1998).

H.F. Fisher, S.K. Saha, and S.J. Maniscalco. Identification and Characterization of Kinetically Competent Carbinolamine and £Iminoglutarate Complexes in the Glutamate Dehydrogenase-Catalyzed Oxidation of L-Glutamate Using a Multiwavelength Transient State Approach, Biochemistry, 37, 14585-14590 (1998).

L.Indyk and H.F. Fisher. Theoretical Aspects of Isothermal Titration Calorimetry. Methods in Enzymology, 295, 350-364 (1998).

H.F. Fisher and J. Tally. Isoergonic Cooperativity¡XA Novel Form of Allostery. Methods in Enzymology, 295, 331-349, (1998).

S.K. Saha, S.J. Maniscalco, and H.F. Fisher. The Use of Multiwavelength Kinetic Analysis Approach to Identify and Characterize Intermediate Complexes in the Reductive Amination Reaction Catalyzed by Bovine Liver Glutamate Dehydrogenase. Biochimica et Biophysica Acta, 1382, 8-12 (1998).

H.F. Fisher and J. Tally. Isoergonic Cooperativity in Glutamate Dehydrogenase Complexes: A New Form of Allostery. Biochemistry, 36 #36, 10807-10810 (1997).

S.K. Saha, S.J. Maniscalco, and H.F. Fisher. Mechanistic Interpretation of Tryptophan Fluorescence Quenching in the Time Courses of Glutamate Dehydrogenase Catalyzed Reaction. Biochemistry, 35, 16483-16488 (1996).

N. Singh, Z. Liu, and H.F. Fisher. The Existence of a Hexameric Intermediate with Molten-Globule-Like Properties in the Thermal Denaturation of Bovine-Liver Glutamate Dehydrogenase. Biophysical Chemistry, 63, 27-36 (1996).

L.Xue, P. Talalay, and A.S. Mildvan, Studies of the Catalytic Mechanism of an Active-Site Mutant (Y14F) of Ą5-3-Ketosteroid Isomerase by Kinetic Deuterium Isotope Effects, Biochemistry, 30:10858, 1991) Critical Reviews in Chemtracts, 6, 69-75 (1996).

S.J. Maniscalco, S.K. Saha, P. Vicedomine, and H.F. Fisher. A Difference in the Sequence of Steps in the Reactions Catalyzed by Two Closely Homologous Forms of Glutamate Dehydrogenase. Biochemistry, 35: 89-94 (1996).

H.F. Fisher and S,K. Saha. The Interpretation of Transient-State Kinetic Isotope Effects. Biochemistry, 35:83-88 (1996).

H.F. Fisher. "Calorimetric Methods for Interpreting Protein-Ligand Interactions." in Methods in Enzymology, Vol. 259, eds. G.K. Ackers and M.L. Johnson, Academic Press, Inc., 194-221, 1995.

S.K. Saha, S.J. Maniscalco, N. Singh, H.F. Fisher. The Demonstration of a Glutamate Dehydrogenase--NADP-L-Glutamate Charge-transfer Complex and Its Location on the Reaction Pathway. J. Biol. Chem. 269:29592-29597 (1994).

S. Pazhanisamy, S.J. Maniscalco, N. Singh, and H.F. Fisher. A Kinetic Mechanism of the Allosteric Control of Enzyme-coenzyme Binding: Glutamate Dehydrogenase-NADPH-Phosphate-Acetate-Hydrogen Ion Interactions. Biochemistry , 33:10381-10385 (1994).

N. Singh, S. Maniscalco, and H.F. Fisher. The Real-Time Resolution of Proton-Related Transient-State Steps in an Enzymatic Reaction. J. Biol. Chem., 268:21-28 (1993)

H.F. Fisher, S. Maniscalco, N. Singh, R.N. Mehrotra, and R. Srinivasan. A Slow Obligatory Proton Release Step Precedes Hydride Transfer in the Liver Glutamate Dehydrogenase Catalytic Mechanism. Biochim. Biophys. Acta, 1119:52-56, 1992.

L Xue, A. Kuliopulos, A.S. Mildvan, and P. Talalay, Catalytic Mechanism of an Active-Site Mutant (D38N) of ƒ´5-3-Ketosteroid Isomerase. Direct Spectroscopic Evidence for Dienol Intermediates, Biochemistry, 30:4991, 1991

H.F. Fisher and N. Singh. Transduction of Enzyme-Ligand Binding Energy into Catalytic Driving Force. FEBS Letters, 294:1-5, 1991.

H.F. Fisher and N. Singh. The Meaning of Interaction Parameters in Two-State Protein Complexes. J. Biol. Phys., 17:213-220, 1990.

H.F. Fisher. (L. Xue, P. Talalay, and A.S. Mildvan, Studies of the Mechanism of the ƒ´5-3-Ketosteroid Isomerase Reaction by Substrate, Solvent, and Combined Kinetic Deuterium Isotope Effects on Wild-Type and Mutant Enzymes, Biochemistry, 29:7491, 1990

H.F. Fisher, S. Maniscalco, N. Singh, S.A. Adediran. Proton/Product Time Course Ratios: A New Approach to Transient-State Kinetic Analysis. J. Biol. Chem., 263:11704-11710, 1988.

R. Srinivasan, T.S. Viswanathan, and H.F. Fisher. Mechanism of Formation of Bound a-Iminoglutarate from a-Ketoglutarate in the Glutamate Dehydrogenase Reaction. J. Biol. Chem., 263:2304-2308, 1988.

H.F. Fisher, S. Pazhanisamy, R.T. Medary. The Anomalous Properties of the Glutamate Dehydrogenase--NADPH--a-Ketoglutarate Complex are not Ascribable to Carbonyl Addition Reactions. J. Biol. Chem., 262:11684-11687, 1987.

H.F. Fisher. "A Unifying Model of the Thermodynamics of Formation of Dehydrogenase-ligand Complexes" in Advances in Enzymology, Vol. 61, ed. A. Meister, John Wiley and Sons, 1-46, 1988.

H.F. Fisher, S. Maniscalco, C. Wolfe, land R. Srinivasan. NADPH-Binding-Induced Proton Ionization as a Cause of Non-Linear Heat Capacity Changes in Glutamate Dehydrogenase. Biochemistry, 25:2910-2915, 1986.

T.S. Viswanathan and H.F. Fisher. Carbonyl Oxygen Exchange Evidence of Imine Formation in Glutamate Dehydrogenase Reaction and Identification of the "Occult Role" of NADPH. Proc. Natl. Acad. Sci., 81:2747-2751, 1984.

H.F. Fisher, A.H. Colen and R.T. Medary. Temperature-Dependent DCp° Generated by a Shift in Equilibrium Between Macrostates of an Enzyme. Nature, 292:271-272, 1981.