Research Interests:

• Biochemistry
• Acetylcholinesterase Studies
• Thrombin Studies

The most fundamental idea pursued in our research program was the origin and evolutionary progress of catalytic acceleration by enzymes. The level of molecular recognition and the catalytic potential of active functional groups in small proteins and serine protease enzymes are probed. Specific targets are the cardiovascular enzymes and the cholinesterases, which were of great scientific and medical interest.

A central question addressed in our studies was the role of short-strong-hydrogen bonds (SSHBs) in catalysis and molecular recognition. A key target of this project is human ?-thrombin inhibited by a variety of small and large peptide inhibitors. The quest for understanding catalytic efficiency included the role of general protein dynamics and their influence on remote-site interactions that are critical in the interaction of enzymes with large protein or peptide natural substrates and other effectors of α-thrombin.  Acetylcholinesterase as an evolutionarily highly developed catalyst was the subject of many investigations aimed at evaluating the importance of optimization of the step for leaving group departure in hydrolytic enzymes and the sensitivity of the catalytic machinery to alterations in the electronic and steric environments of substrates and inhibitors.

Our studies span the fields of physical organic chemistry (particularly isotope effects), biochemistry (particularly mechanistic enzymology) and, occasionally, organic synthesis. Experimental tools include stopped-flow kinetics, spectroscopic, fluorometric and electro-chemical methods. High-resolution 1H NMR, especially its applications to the characterization of deshielded SSHBs, was used in collaboration. Complementary structural information was obtained with molecular modeling of macromolecule-small molecule interactions by computer graphics combined with various energy minimizations and molecular dynamics calculations.  Funding of these efforts were provided by the NIH, American Heart Association and DOD.

Selected Publications

I.M. Kovach, Proton Bridging in Catalysis by and Inhibition of Serine Proteases of the Blood CascadeSystem, Life, 11(5), 396 (2021). https://doi.org/10.3390/life11050396 - retrieved 27 Apr 2021.

I. M. Kovach,* L. Kakalis, F. Jordan and D. Zhang, Proton Bridging in the Interactions of Thrombin with Hirudin and Its Mimics, Biochemistry 52, 2472-2481 (2013). DOI: 10.1021/bi301625a -  retrieved 10 May 2021. 

C. Furman, J. Mehla, N. Ananthaswamy, N. Arya, B. Kulesh, I. M. Kovach, S.V. Ambudkar, J. Golin. The Deviant ATP-binding Site of the Multidrug Efflux Pump Pdr5 Plays an Active Role in the Transport Cycle, J. Biol. Chem. 288, 30420-30431 (2013). DOI: 10.1074/jbc.M113.494682 -  retrieved 10 May 2021. 

I. M. Kovach,  P. Kelley, C. Eddy, F. Jordan, and  A. Baykal, Proton Bridging in the Interaction of Thrombin with Small Inhibitors.  Biochemistry 48, 7296-7304 (2009).

D. Zhang and I. M. Kovach and J. P. Sheehy Locating the Rate-Determining Step for Three-Step Hydrolase-Catalyzed Reactions with  DYNAFIT. Biochimica at Biophysica Acta: Proteins and Proteomics 1784, 827-833, (2008).

D. Zhang and I. M. Kovach   Deuterium Solvent Isotope Effect and Proton Inventory Studies of Factor Xa-Catalyzed Reactions. Biochemistry 45, 14175-14182, (2006).

D. Zhang and I. M. Kovach Full and Partial Deuterium Solvent Isotope Effect Studies of a-Thrombin-Catalyzed Reactions of Natural Substrates,  J. Am. Chem. Soc. 127, 3760-3766 (2005).

E. J. Enyedy and I. M. Kovach  Proton Inventory Studies of a-Thrombin-catalyzed Reactions of Substrates with Selected P and P’ Sites. J. Am. Chem. Soc. 126,  6017-6024 (2004).

I. M. Kovach  Stereochemistry and Secondary Reactions in the Irreversible Inhibition of Serine Hydrolases by Organophosphorus Compounds. J. Physical Organic Chemistry 17, 602-614 (2004) invited.

A.S. Mildvan, M.A. Massiah, T.K. Harris, G.T. Marks, D.H. T. Harrison, C. Viragh, P. M. Reddy and I. M. Kovach  Short Strong Hydrogen Bonds on Enzymes: NMR and Mechanistic Studies. J. Mol. Structure 163-175, (2002).

P. M. Reddy and I. M. Kovach  Synthesis of chiral methyl 4-nitrophenyl alkylphosphonothioates: BF3-Et2O-catalyzed alcoholysis of phosphonamidothioates. Tetrahedron Letters 43, 4063-4066 (2002).

P. M. Reddy, C. Viragh and I. M. Kovach  Synthesis of Phenyl or 4-Nitrophenyl Methyl  b-Ketophosphonate Esters. Phosphorus Sulfur and Silicon 177, 1-4, (2002).

A.S. Mildvan, M.A. Massiah, T.K. Harris, G.T. Marks, D.H. T. Harrison, C. Viragh, P. M. Reddy and I. M. Kovach   Short Strong Hydrogen Bonds on Enzymes: NMR and Mechanistic Studies. J. Mol. Structure (2002).

I. M. Kovach   Inhibition of Serine Hydrolases by Organophosphorus Compounds. Biokemia XXV, 74-80 (Hungarian invited review) 2001.

I. J. Enyedy and I. M. Kovach, A. Bencsura, A Molecular Dynamics Study of Active-site Interactions with Tetracoordinate Transients in Acetylcholinesterase and its Mutants. Biochemical J. 353, 645-653 (2001).

M. A. Massiah, C. Viragh, P. M. Reddy, I. M. Kovach, J. Johnson, T. L. Rosenberry and A. S. Mildvan, Short, Strong Hydrogen Bonds at the Active Site of Human Acetylcholinesterase: 1 H-NMR Studies.  Biochemistry 40, 5682-5690 (2001).

C. Viragh, T. K. Harris, P. M. Reddy, M. A. Massiah, A. S. Mildvan and I. M. Kovach, NMR Evidence for a Short, Strong Hydrogen Bond in the Mechanism of a Cholinesterase. Biochemistry 39, 16200-16205 (2000).  

E. Enyedy and I. M. Kovach, Reversible Modulation of Human Factor Xa  Activity by Phosphonate Ester: Media Effects. Bioorg. Med. Chem. 8, 549-556 (2000).

C. Viragh, I. M. Kovach and Lewis Pannell,  Small Molecular Products of Dealkylation in Soman-inhibited Electric Eel Acetylcholinesterase. Biochemistry 38, 9557 (1999).

I. M. Kovach, Ligand and Active-Site Dependent P-O Versus C-O Bond Cleavage in Organophosphorus Adducts of Serine Hydrolases. Phosphorus, Sulfur and Silicon, 144-146, 537-540 (1999).

A. Saxena, C. Viragh, D. S. Frazier, I. M. Kovach, D. M. Maxwell,  O. Lockridge and B. P. Doctor, The pH Dependence of Dealkylation in Soman-inhibited Cholinesterases and Their Mutants: Further Evidence for a Push-Pull Mechanism. Biochemistry 37, 15086-15096 (1998).

I. J. Enyedy, I. M. Kovach and B. R. Brooks, Alternate Pathways for Acetic Acid and Acetate Ion Release from Acetylcholinesterase: A Molecular Dynamics Study.  J. Am. Chem. Soc. 120, 8043-8050 (1998).

I. M. Kovach and E. Enyedy, Active-site-dependent Elimination of 4-Nitrophenol from 4-Nitrophenyl Alkylphosphonyl Serine Protease Adducts. J. Am. Chem. Soc. 120, 258-263  (1998).

I. M. Kovach, R. Akhmetshin, I. J. Enyedy and C. Viragh A Selfconsistent Mechanism for Dealkylation in Soman-inhibited Acetylcholinesterase. Biochemical J. 324, 995-996 (1997).

C. Viragh, R. Akhmetshin, I. M. Kovach, and C. Broomfield, Unique Push-Pull Mechanism of Ageing in Soman-inhibited Acetylcholinesterase. Biochemistry 36, 8243-8252 (1997).

E. Enyedy and I. M. Kovach, Modulation of the Activity of Human α-Thrombin with Phosphonate Ester Inhibitors.   Bioorg. Med. Chem. 5, 1531-1541, (1997).

A. Bencsura, I. Enyedy and I. M. Kovach, Probing the Active Site of Acetylcholinesterase by Molecular Dynamics of its Phosphonate Ester Adducts. J. Am. Chem. Soc. 118, 8531-8541 (1996).

I. Enyedy, A. Bencsura and I. M. Kovach, Interactions in Tetravalent and Pentavalent Phosphonate Esters of Ser at the Active Site of Serine Enzymes.  Phosphorus, Sulfur, and Silicon 109-110, 249-252 (1996).

Q. Zhao and I. M. Kovach, Reversible Modification of Tissue-type Plasminogen Activator by Methylphosphonate Esters . Bioorg. Med. Chem 4, 523-529 (1996).

A. Bencsura, I. Enyedy and I. M. Kovach, Origins and Diversity of the Aging Reaction in Phosphonate Adducts of Serine Hydrolase Enzymes: What  Characteristics of the Active Site Do They Probe? Biochemistry 34, 8989-8999 (1995).

I. M. Kovach, N. Qian and A. Bencsura, Efficient Product Clearance through Exit Channels in Substrate Hydrolysis by Acetylcholinesterase. FEBS Lett. 349, 60-64 (1994).

A. Saxena, N. Qian, I. M. Kovach, A. P. Kozikowski, Y. P. Pang, D. C. Vellom, Z. Radic, D. Quinn, P. Taylor, and B. P. Doctor, Identification of Amino Acid Residues Involved in the Binding of Huperzine-A to Cholinesterases. Protein Sci., 1770-1778 (1994).

Q. Zhao, I.M. Kovach, A. Bencsura and  A. Papathanassiu, Enantioselective and Reversible Inhibition of Trypsin and α-Chymotrypsin by Phosphonate Esters. Biochemistry 33, 8128 (1994).

N. Qian and I. M. Kovach, Key Active Site Residues in the Inhibition of Acetylcholinesterases by Soman.  FEBS Lett. 336, 263 (1993).

I. M. Kovach, Q. Zhao, M. Keane and R. Reyes, Rate-Determining Carbonyl Hydration in the Intramolecular Hydrolysis of Phenacyl Phosphonate Esters: Isotopic Probes and Activation Parameters. J. Am. Chem. Soc.. 115, 10471 (1993).

I. M. Kovach, A.J. Bennet, J.A. Bibbs, and Q. Zhao, Nucleophilic and Protolytic Catalysis of Phosphonate Ester Hydrolysis: Isotope Effects and Activation Parameters. J. Am. Chem. Soc.  115, 5138 (1993).

I. M. Kovach Solution Dynamics of Phosphonate Ester Hydrolysis.  Phosphorus, Sulfur and Silicon 75, 131 (1993).

I. M. Kovach, L. McKay and D. Vander Velde, Diastereomeric Phosphonate Ester Adducts of Chymotrypsin: 31P-NMR  Measurements. Chirality 5, 143 (1993).

I. M. Kovach and L. McKay  Reversible Modulation of Serine Protease Activity by Phosphonate Esters. Bioorg. Med. Chem. Lett. 2, 1735 (1992).

I. M. Kovach and D. Huhta  Comparative Study of the Charge Distribution in Tetravalent Carbonyl Transients and Organophosphorus Adducts of Trypsin. J. Mol. Struct. (Theochem 79) 233, 335 (1991).

I. M. Kovach, Competitive Irreversible Inhibition of Enzymes in the Presence of a Substrate: Scope and Limitations. J. Enzyme Inhibit. 4, 201 (1991).

I. M. Kovach, D. Huhta and S. Baptist  Active Site Interactions in Hydrated Trypsin-Organophosphate Adducts: A YETI Molecular Mechanics Study.  J. Mol. Struct. (Theochem 72) 226, 99 (1991).

I. M. Kovach, Son Do and R.L. Schowen β-Secondary Deuterium Isotope Effect and Solvent Isotope Effects in Catalysis by Subtilisin BPN'. J. Phys. Org. Chem. 3, 260  (1990).

I. M. Kovach and A.J. Bennet Comparative Study of Nucleophilic and Enzymic Reactions of 2-Propyl Methylphosphonate Derivatives. Phosphorus, Sulfur and Silicon 51/52, 51 (1990).

Some Invited Presentations

“Short Strong Hydrogen Bonds at the Active Site of Cholinesterases” I. M. Kovach, Gordon Research Conference on Isotopes in Biological and Chemical Sciences, Ventura, CA, February 17-22, 2002.

“Short Strong Hydrogen Bonds at the Active Site of Cholinesterases: 1H-NMR Studies”  I. M. Kovach, C. Viragh, P. M. Reddy, M. A. Massiah, A. S. Mildvan, J. Johnson and T. L. Rosenberry, VIIth International Meeting on Cholinesterases, Pucon, Chile, November 8-12, 2002.

“Protonic Participation in the Rate-determining Step for the Hydrolysis of Fluorogenic Substrates Catalyzed by Hemostatic and Thrombolytic Enzymes.” I. M. Kovach, E. J. Enyedy and R. A. Akhmetshin, International  Isotope Effects Conference an EUCHEM Conference, Uppsala, Sweden, June 22-27, 2003.

"Protonic Participation in Hemostatic and Thrombolytic Enzyme-catalyzed Reactions." I. M. Kovach, E.J. Enyedy and R.A. Akhmetshin, XVth  International Conference on Horizons in Hydrogen Bond Research, Freie Universitat Berlin, Germany, September 16-21, 2003.

“Full and Partial Deuterium Solvent Isotope Effect Studies of a-Thrombin-Catalyzed Reactions of Natural Substrates” D. Zhang and I. M. Kovach,  Isotopes 2005 Bath: An international conference, The University of Bath, UK, June 27- July 1, 2005.

"Short Strong Hydrogen Bonds in Support of the Inhibition of Human a-Thrombin." I. M. Kovach, Gordon Research Conference on Isotopes in Biological and Chemical Sciences, Galveston, TX, February 14-19, 2010.

Patents

I.M. Kovach, Temporary Inactivation of Serine Hydrolases Using Nitrophenyl Phenacyl Phosphonates.  US patent 5,281,523 Ser.# 851,187, January 25, 1994. US patent 5,324,815 Ser.# 850,112, May 24, 1994

Awards

Fogarty Fellowship, Hungarian National Academy of Sciences, Institute of Enzymology, Budapest Hungary. (Summer 1983)

Senior Faculty Research Associateship in the Navy-ASEE program. Laboratory for the Structure of Matter, Naval Research Laboratory, Washington , DC (Summer 1990)

NSF Career Advancement Award for Women (May 1990)

American Heart Association, Nation's Capital Affiliate 1992 Heart Ball Research Award (April 1992)

NIH/NIA Individual National Research Service Award, Laboratory of Structural Biology, DCRT, NIH, Bethesda, MD (January 1996)

NRC Senior Research Associateship, Division of Biochemistry, Walter Reed Army Institute of Research, Washington, DC. (March 1996)

Editor and Member of the Editorial Board of the Biochemical Journal. (1999-2009)

Senior Fulbright  lecturing award, 2003-2004 host institute Department of Biochemistry at Eotvos Lorand University. (2003-2004)