The traditional paradigm for the function of enzymes invokes the combined action of active site amino acid side chains in concert with small, exogenous molecules (metal ions or organic cofactors) that bind to the active site. In recent years, post-translational modification of proteins has been shown to extend this paradigm to include new active site functionalities that arise from the modification of the standard 20 amino acids.

This laboratory has been intimately involved in the discovery and characterization of a family of quino-cofactors that are derived from peptide and protein-bound aromatic residues, tyrosine and tryptophan. These include trihydroxyphenylalanine quinone (LTQ), a lysine cross-linked tryptophan quinone (TTQ and CTQ, respectively). Additionally, a free standing quino-cofactor has been characterized that arises from the cross-linking of a peptide-bound glutamate to tyrosine to produce pyrroloquinoline quinone (PQQ).

Proteins that contain the TPQ cofactor (called the copper amine oxidases) have received the most intensive study, with their exceptional property of being able to catalyze two distinctive reactions: the generation of the TPQ cofactor and its subsequent use in catalysis. Studies in progress indicate that the active site structures for both the precursor and active form of the copper amine oxidases are almost superimposable. This raises the provocative question of how a single folded protein can catalyze disparate reactions while maintaining tight control over the individual reactions. Very recent results implicate a conserved active site tyrosine that when mutated to phenylalanine subverts cofactor biogenesis, leading instead to the insertion of three hydroperoxide groups into the aryl ring of the cofactor precursor turosine! This result, undergoing further study, is just one example of the surprises that continue to evolve from studies of the TPQ-containing proteins.

In addition to their unusual catalytic properties, the physiological role of the mammalian copper amine oxidases has remained an enigma. The human genome annotates three open reading frames for these copper amine oxidases: AOC-1, AOC-2, and AOC-3. The isozyme AOC-3 has received the greatest attention, residing on the outer plasma membrane of endothelial tissue that AOC-3 plays a role in leukocyte recruitment to the endothelial tissue that lines the vasculature. We are focused on the adipocyte-associated AOC-3, in particular, the role of the enzymatically produced hydrogen peroxide in cell signalling. Many experiments are in progress that include the monitoring of intracellular phosphorylation cascades, extracellular cytokine release and changes in the levels of intracellular RNA. Data collected to date indicate that the activity of AOC-3 is linked to the central role of the adipocyte in cellular immunity and in an accompanying energy homeostasis.

In contrast to TPQ, the free standing quino-cofactor, PQQ, requires the participation of six open reading frames that, collectively, catalyze a cross-linking reaction between a glutamate and a tyrosine side chain, an overall 12-electron oxidation that also involves an incorporation of molecular oxygen and, ultimately, hydrolysis at four positions withi the precursor polypeptide chain. Each of the requisite gene products for biogenesis is undergoing intensive investigation within the laboratory.

 

A very large number of key biological functions involve the use of molecular oxygen. Given the inherent toxicity of activated oxygen species, the success of aerobic organisms is intimately tied to the concomitant emergence of strategies that allow molecular oxygen to be activated while limiting oxidative damage to the catalysts themselves. Patterns have begun to emerge regarding the nature of biological dioxygen reactivity. For example, in the glucose oxidase reaction, we have shown how a single active site charge can be used to reduce the barrier for O2 reduction in the absence of significant accumulation of the resulting reactive oxygen species. Another feature that has emerged from recent studies is the participation of discrete channels and pockets in enzymes that can control and direct O2 binding and reactivity.

One of the enzyme systems that has been studied for many years in this laboratory is dopamine beta-monooxygenase (DbM), which catalyzes the formation of the hormone/neurotransmitter norepinephrine from dopamine within the chromaffin and synaptic storage vesicles. In contrast to the TPQ-containing amine oxidases, DbM contains only copper as its cofactor. The role of copper in oxygen/substrate activation and the nature of reactive oxygen intermediates has been quite puzzling, given that DbM catalyzes a hydroxylation reaction at a solvent interface and that this involves the transfer of an electron ca. 11 angstrom across an aqueous interface.

Over the years, we have developed methodologies for distinguishing between O-16 and )-18 reactivity in oxygen-dependent reactions. Our recent probes of DbM have implicated an initially-unanticipated mechanism in which a copper-superoxo- species initiates C-H activation. We have been applying these methodologies to a range of oxygen-dependent enzymes, which include, in addtion to DbM and glucose oxidase, lipoxygenase (an iron protein), peptide amidating enzyme (a copper protein), tyrosine hydroxylase (a pterin/iron system, catalyzing dopa formation from tyrosine), cytochrome P-450 (a heme/iron system), and ACC oxidase (a non-heme iron enzyme that produces the plant hormone ethylene from aminocyclopropane carboxylate. Our long-range goal is to be able to systematize the strategies that enzymes use to achieve the efficient activation of dioxygen.

 

 

Over the course of our investigations of enzyme-catalyzed redox reactions, a number of anomalies have suggested that quantum chemistry may dominate hydrogen transfer at enzyme active sites. Extensive work from this laboratory (as well as the work of others) has now shown that hydrogen tunneling occurs in virtually every C-H cleavage that focuses on mtions within the protein backbone as the origin of the reaction barrier. The effects of alteratin in both substrate and protein structure, with the latter including a comparison of mesophilic enzymes to their thermophilic and psychrophilic homologs,m is shedding light on how the protein facilitates wave function overlap between the donor and acceptor atoms. Protein dynamics plays a key role in this effect through an intial pre-organization, to select the subset of protein conformations that will be optimal for catalysis, and a subsequent reorganization that includes the tuning of the active site electrostatics and the distance between the reactants. There is a very interesting inverse correlation between preorganization and reorganization, with the active site being less dynamical whenn the pre-organization has been optimized. These results are changing our view of enzyme catalysis, moving us beyond the static, transition state stabilization model to one that embraces the importance of barrier width and protein dynamics.