The overall aim is to understand how the environment of an enzyme active site controls the reactivity of a catalytic center containing two metal ions connected by a bridging ligand. Bridged bimetallic centers appear in hundreds of different enzymes catalyzing dozens of different reactions including the degradation and synthesis of DNA, RNA and phospholipids; hydride ion transfer; phosphoryl group transfer; the hydrolysis of sugars; and the covalent modification and N-terminal processing of polypeptides. Yet why two metal ions are employed, and how they cooperate in catalysis is not understood.
The specific aims focus on: i) how the metal ions cooperate, ii) why particular metal/ligand combinations are most effective in catalyzing particular types of reactions, and iii) how the rest of the active site participates in, and helps to direct the chemistry of, catalysis. Four representative enzymes have been selected for this study. In xylose isomerase a bridged Mg2+-Mg2+ center converts glucose to fructose, a reaction of enormous commercial importance. Aminopeptidase uses a Zn2+-Zn2+ center in an exopeptidase reaction; members of this enzyme superfamily are targets for antiangiogenic cancer drugs. DRAG uses Mg2+-Mg2+ to catalyze the hydrolysis of ADP-ribose from a covalently-modified protein. And the immunosuppressant target calcineurin uses an Fe3+-Zn2+ center in its role as an essential protein Ser/Thr phosphatase in signal transduction. Our experimental plan is guided by the hypothesis that bridged bimetallic enzymes use the metal ion Lewis acidities in their binuclear sites to: i) bind and position substrate, ii) bind and activate a water molecule to yield an active site hydroxide nucleophile, and iii) act as a """"""""superelectrophile"""""""" to polarize a chemical bond on the substrate and thereby promote formation of the transition state of the catalytic reaction. We will employ a variety of methods, including ones we have developed ourselves. These techniques include protein crystallography (conventional, ultra-high resolution, and time-resolved), neutron Laue crystallography, enzyme kinetics, spectroscopy (in collaboration), quantum mechanics/molecular mechanics calculations, and site-directed mutagenesis.
|Deshpande, Aditi R; Pochapsky, Thomas C; Ringe, Dagmar (2017) The Metal Drives the Chemistry: Dual Functions of Acireductone Dioxygenase. Chem Rev 117:10474-10501|
|Deshpande, Aditi R; Wagenpfeil, Karina; Pochapsky, Thomas C et al. (2016) Metal-Dependent Function of a Mammalian Acireductone Dioxygenase. Biochemistry 55:1398-407|
|Huang, Yu-Hwa; Zhu, Chen; Kondo, Yasuyuki et al. (2015) CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Nature 517:386-90|
|Liu, Ce Feng; Liu, Dali; Momb, Jessica et al. (2013) A phenylalanine clamp controls substrate specificity in the quorum-quenching metallo-?-lactonase from Bacillus thuringiensis. Biochemistry 52:1603-10|
|Auclair, Jared R; Somasundaran, Mohan; Green, Karin M et al. (2012) Mass spectrometry tools for analysis of intermolecular interactions. Methods Mol Biol 896:387-98|
|Somarowthu, Srinivas; Brodkin, Heather R; D'Aquino, J Alejandro et al. (2011) A tale of two isomerases: compact versus extended active sites in ketosteroid isomerase and phosphoglucose isomerase. Biochemistry 50:9283-95|
|Lazar, Louis M; Fisher, S Zoe; Moulin, Aaron G et al. (2011) Time-of-flight neutron diffraction study of bovine ?-chymotrypsin at the Protein Crystallography Station. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:587-90|
|Liu, Dali; Momb, Jessica; Thomas, Pei W et al. (2008) Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis. 1. Product-bound structures. Biochemistry 47:7706-14|
|Momb, Jessica; Wang, Canhui; Liu, Dali et al. (2008) Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis. 2. Substrate modeling and active site mutations. Biochemistry 47:7715-25|
|Munih, Petra; Moulin, Aaron; Stamper, Carin C et al. (2007) X-ray crystallographic characterization of the Co(II)-substituted Tris-bound form of the aminopeptidase from Aeromonas proteolytica. J Inorg Biochem 101:1099-107|
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