The Use of Isotope Effects to Study Dopamine β-Monooxygenase Catalysis: the Work of Judith P. Klinman

Abstract

Deduction of the Kinetic Mechanism in Multisubstrate Enzyme Reactions from Tritium Isotope Effects. Application to Dopamine β-Hydroxylase

(Klinman, J. P., Humphries, H., and Voet, J. G. (1980) J. Biol. Chem. 255, 11648–11651)

Use of Isotope Effects to Characterize Intermediates in the Mechanism-based Inactivation of Dopamine β-Monooxygenase by β-Chlorophenethylamine

(Bossard, M. J., and Klinman, J. P. (1990) J. Biol. Chem. 265, 5640–5647)

Judith Klinman was born Judith Pollock in 1941 in Philadelphia, Pennsylvania. Early on, she realized she was interested in science and decided to go to the University of Pennsylvania to study chemistry. However, her parents wanted her to become a medical technologist and forget about science. She eventually got them to agree to let her go if she got a scholarship; she graduated in 1962 with an A.B. She then decided to go to graduate school at New York University, but moved back to Penn after a year. Working with Edward R. Thornton, she completed her Ph.D. in 3 years, publishing a thesis titled “A Kinetic Study of the Hydrolysis and Imidazole-catalyzed Hydrolysis of Substituted Benzoyl Imidazole in Light and Heavy Water.”

Judith P. Klinman

After graduating in 1966, Klinman first carried out postdoctoral research with David Samuel at the Weizmann Institute of Science in Israel and later with Journal of Biological Chemistry (JBC) Classic author Irwin Rose (1) at the Institute for Cancer Research in Philadelphia. Returning to the United States in 1968, she joined the Institute for Cancer Research in Philadelphia, where she was a research scientist for 10 years. In 1978 she became the first woman professor in the chemistry department of the University of California, Berkeley, where she continues to do research today as Professor of Chemistry and Molecular and Cell Biology in the Department of Chemistry.

Throughout her research career, Klinman has contributed extensively to the understanding of the fundamental properties that underlie enzyme catalysis. Early in her career, she developed the application of kinetic isotope effects to the study of enzyme catalysis, showing how these probes can be used to uncover chemical steps, to determine kinetic order, and to obtain substrate dissociation constants. The two JBC Classics reprinted here stem from her use of isotope effects to isolate the chemical steps involved in the dopamine β-monooxygenase-catalyzed conversion of dopamine and oxygen to norepinephrine and water.

In 1965, JBC Classic author Seymour Kaufman (2) suggested that oxygen binding precedes the addition of substrate in dopamine β-hydroxylase (now called dopamine β-monooxygenase) (3). However, confirmation of this hypothesis was hard to do using classical methods such as product and dead-end inhibition studies and equilibrium exchange techniques because of the apparent reversibility of the chemical step and the fact that one of the reaction products was water. In the first Classic, Klinman was able to disprove this hypothesis and use the sensitivity of kinetic tritium isotope effects to changes in oxygen concentrations in the reaction to provide unequivocal evidence for a random order of addition of dopamine and oxygen to dopamine β-hydroxylase.

In the second Classic, Klinman uses isotope effects to study the inhibition of dopamine β-monooxygenase by β-chlorophenethylamine. Previously, she had postulated an inhibition mechanism in which bound α-aminoacetophenone was generated followed by an intramolecular redox reaction to yield a ketone-derived radical cation as the inhibitory species (4). However, she was unable to determine whether inhibition by α-aminoacetophenone occurred at the reductant- or substrate-binding site and was unable to provide evidence of keto-enol tautomerization of α-aminoacetophenone at the active site. Both of these questions were addressed using kinetic isotope effects. As reported in the JBC Classic, she showed that α-aminoacetophenone acts at the substrate-binding site and that there are two isotope-sensitive steps in β-chlorophenethylamine inactivation, with the second step attributed to an isotope-sensitive partitioning of the bound enol of α-aminoacetophenone between reketonization and oxidation.

Over the years, Klinman continued her investigations into enzyme catalysis. In 1990 she demonstrated the presence of the neurotoxin, 6-hydroxydopaquinone (TPQ), at the active site of a copper-containing amine oxidase from bovine plasma, overcoming years of incorrect speculation regarding the nature of the active site structure and opening up the currently active field of protein-derived cofactors. Subsequent work from her group showed that the extracellular protein lysyl oxidase, responsible for collagen and elastin cross-linking, contains a lysine cross-linked variant of TPQ. Since the 1990s, Klinman's kinetic studies of enzyme reactions have demonstrated anomalies that implicate quantum mechanical hydrogen tunneling in enzyme-catalyzed hydrogen activation reactions. In recent years she has developed a unique set of experimental probes for determining the mechanism of oxygen activation. These probes are beginning to shed light on how proteins can reductively activate O₂ to free radical intermediates, while avoiding oxidative damage to themselves.

In addition to being the first woman faculty member in the physical sciences at the University of California, Berkeley, Klinman was the first woman Chair of the Department of Chemistry from 2000 to 2003. During her tenure at Berkeley she has been a Chancellor's Professor, Guggenheim Fellow, and Miller Fellow. She was elected to the National Academy of Sciences (1994), the American Academy of Arts and Sciences (1993), and the American Philosophical Society (2001) and has received the Repligen Award (1994) and the Remsen Award (2005) from the American Chemical Society and the Merck Award from the American Society for Biochemistry and Molecular Biology (2007). Klinman was also President of the American Society for Biochemistry and Molecular Biology in 1998 and served on the editorial board of the Journal of Biological Chemistry from 1979 to 1984.