Judith Klinman

Judith P. Klinman

Professor Emerita of Biochemistry, Biophysics and Structural Biology

Env Full Directory Information

Research Interests

The overall focus of our research is the determination of fundamental principles of catalytic and regulatory mechanism in enzyme-catalyzed reactions. Our experimental approach is broadly based and combines kinetic, spectroscopic, stereochemical, and molecular biological techniques.

Current Projects

Protein and peptide- derived cofactors. Work over the last decade has forced us to expand our definition of enzymatic cofactors to include structures that are derived from the protein side chains themselves. Early studies by others had implicated simple radicals (e.g., the tyrosyl and glycyl radicals), whereas more recent work, (including much from this laboratory) indicates complex structures that require novel chemical pathways. We are currently focused on four distinct structures that are derived from protein- or peptide-bound tyrosine side chains: the 2,4,5-trihydroxyphenylalanyl quinone (TPQ) found in the ubiquitous copper amine oxidases, the lysine tyrosyl quinone (LTQ) found in the mammalian lysyl oxidases, the cysteine tyrosyl radical (CTR) in the fungal galactose oxidase, and pyrroloquinoline quinone (PQQ), an essential vitamin for certain bacteria. Studies of the biogenesis of each of these structures require the expression of a precursor protein (or peptide in the case of PQQ), together with the establishment of conditions for monitoring the biogenetic processes. In the case of TPQ, LTQ and CTR all evidence indicates that these are produced by self-processing pathways that occur in the absence of exogenous protein factors, whereas PQQ requires six gene products. Both TPQ and LTQ were discovered in this laboratory and studies on the formation of TPQ are the most advanced, offering valuable protocols for studies of the other systems. In many instances, site specific mutagenesis has proven invaluable, leading to an accumulation of biogenesis intermediates that can be characterized by a variety of spectroscopic techniques. With regard to the function of these intriguing cofactors, recent studies of the mammalian TPQ-containing copper amine oxidases (found on the outer surface of the endothelium and adipocyte) suggest a role for enzymatically-produced hydrogen peroxide in cell signaling. Many experiments are either in progress or planned, to probe these provocative preliminary findings.

Copper- and iron-containing monooxygenases. A very large number of key biological functions involve the use of molecular oxygen. For example, dopamine beta-monooxygenase catalyzes the formation of the hormone/neurotransmitter norepinephrine from dopamine. The enzyme is compartmentalized to either chromaffin vesicles in the adrenal gland or synaptic vesicles in the sympathetic nervous system. In contrast to amine oxidases, dopamine beta-monooxyegnase contains only copper as a cofactor. Key mechanistic questions concern the role of copper in oxygen/substrate activation and the nature of reactive oxygen intermediates. We have developed a set of protocols to study O2 activation which includes distinguishing between O-16 and O-18 reactivity. We are now applying our unique methodology to a range of O2-dependent enzymes, which include dopamine beta-monooxygenase, lipoxygenase (an iron protein), peptide amidation enzyme (a copper protein), tyrosine hydroxylase (a pterin/iron system, catalyzing dopa formation from tyrosine), glucose oxidase (a flavo-protein) and cytochrome P-450 (a heme/iron system). Patterns have begun to emerge regarding dioxygen reactivity. Our goal is to systematize biological systems with regard to their mechanism of dioxygen activation.

Nuclear tunneling in enzyme reactions. Over the course of our investigations of enzyme-catalyzed redox reactions, a number of anomalies had arisen which are incompatible with classical views of catalysis. In particular, these anomalies have suggested that quantum mechanical events may play a significant role in hydrogen transfer events at enzyme active sites. During the last several years, we have expanded the database for proteins that use tunneling as part of their catalytic strategy. Virtually every system examined thus far has shown some evidence for tunneling with the amount of tunneling varying from moderate to extreme. The effect of alteration in both substrate and protein structure is under investigation, with the goal of understanding the effect of changes in protein motion and active site geometry on tunneling. We have many interesting and surprising results that include the findings (i) that single active site residue can control tunneling, and (ii) that thermophilic proteins utilize hydrogen tunneling even at the elevated temperatures of their optimal function.

Selected Publications

Oxygen-18 Isotope Effects as a Probe of Enzymatic Activation of Molecular Oxygen, [J.P. Roth and J.P. Klinman (2006) in Isotope Effects in Chemistry and Biology, (A. Kohen and H. Limbach, eds.) Taylor and Francis Inc., Boca Raton, FL, pp 645-669]

The Copper Enzyme Family of Dopamine beta-Monooxygenase and Peptidylglycine alpha-Hydroxylating Monooxygenase: Resolving the Chemical Pathway for Substrate Hydroxylation, [J.P. Klinman (2006) J. Biol. Chem. 281, 3013-3016]

Modeling Temperature Dependent Kinetic Isotope Effects for Hydrogen Transfer in a Series of Soybean Lipoxygenase Mutants: The Effect of Anharmonicity Upon Transfer Distance, [M. Meyer and J.P. Klinman (2005) Chem. Phys. 319, 283-296]

Mechanism of Post-Translational Quinone Formation in Copper Amine Oxidases and Its Relationship to the Catalytic Turnover. [J.L. DuBois amd J.P. Klinman (2005) Arch. Biochem. Biophys. 433, 255-265]

Thermal Activated Protein Mobility and its Correlation with Catalysis in Thermophilic Alcohol Dehydrogenase, [Z.-X. Liang, T. Lee, K.A. Resing, N.G. Ahn and J.P. Klinman (2004) Proc. Natl. Acad. Sci. USA , 9556-9561]

Quinone Biogenesis: Structure and Mechanism of PqqC, the Final Catalyst in the Production of Pyrroloquinoline Quinone, [O.T. Magnusson, H. Toyama, M. Saeki, A. Rojas, J.C. Reed, R.C. Liddington, J.P. Klinman and R. Schwarzenbacher (2004) Proc. Natl. Acad. Sci. USA , 7913-7918]

Evidence that Dioxygen and Substrate Activation are Tightly Coupled in DβM: Implications for Reactive Oxygen Species, [J.P. Evans, K. Ahn and J.P. Klinman (2003) J. Biol. Chem . , 49691-49698]

Temperature-Dependent Isotope Effects in Soybean Lipoxygenase-1: Correlating Hydrogen Tunneling with Protein Dynamics, [M.J. Knapp, K. Rickert and J.P. Klinman (2002) J. Am. Chem. Soc. 124, 3865-3874]

Investigation of Spectroscopic Intermediates during Copper-Binding and TPQ Formation in Wild-Type and Active Site Mutants of a Copper-Containing Amine Oxidase from Yeast. [J. Dove, B. Schwartz, N. Williams, and J. P. Klinman (2000) Biochemistry 39, 3690-3698]

Life As Aerobes: Are There Simple Rules for Activation of Dioxygen by Enzymes? [J. P. Klinman (2000) J. Biol. Inorg. Chem. 6, 1-13]

Enzyme Dynamics and Hydrogen Tunneling in a Thermophilic Dehydrogenase. [A. Kohen, R. Cannio, S. Bartolucci, and J. P. Klinman (1999) Nature 399, 496-499]

Last Updated 2007-08-27