Dr. Ah-Lim Tsai, Professor

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structure/function and reaction mechanism of important metalloproteins

Increasing number of hemeproteins has been found to play key roles in signal sensing/transduction, oxidation-reduction, gas transport and storage, energy conservation and gene regulation. Elucidation of the basic reaction mechanism of these metalloproteins will provide fundamental insights into their physiological functions, pathological roles and is also useful for development of effective therapeutic agents.

Several heme-containing proteins that control key biosynthetic pathways of prostaglandins (PGs) and nitric oxide (NO) are under active investigation in my laboratory. Prostaglandin H synthase (PGHS or COX), the target of aspirin and other nonsteroidal anti-inflammatory agents (NSAIDs), catalyze the conversion of arachidonic acid to prostaglandin G2 and H2 (PGG2 and PGH2), both are active vasoconstrictor and aggregator of platelets. In addition to the substantial pharmacological interest, the enzymatic catalysis of PGHS is unique at three different aspects: one enzyme has two enzyme activities (peroxidase and cyclooxygenase); catalytic turnover is both self-propagating and self-inactivating. Our center theme is to unveil the coupling mechanism of both enzyme activities, to locate the key intermediate(s) that lead to the self-propagation of the COX activity and the self-inactivation of PGHS. Both PGHS-1 and ­2 isozymes obtained from native source or as recombinant protein are carefully studied using various spectroscopic and kinetic methods to define the structure of key intermediates and the temporal relationship among different intermediates transiently formed during catalysis. I have been collaborating with Dr. Richard J. Kulmacz on both PGHS isozymes for more than 25 years and more than a decade with Drs. Wilfred van der Donk at University of Illinois at Urbana/Champaign and Gary J. Gerfen at Yeshiva University, New York. Recent focus is on the role of oxygen in controlling the dynamics of COX radical intermediates, in peroxide- and AA-induced inactivation and in the catalysis of ferrous PGHS. In collaboration with Dr. Lee-Ho Wang in Hematology Division, we also investigate catalytic mechanisms of the downstream enzymes: thromboxane synthase, which converts PGH2 to thromboxane A2, a most potent native vasoconstrictor and agonist for platelet aggregation; Prostacyclin synthase, using the same substrate PGH2, product of PGHS, but produces a hormonal product exactly antagonizing the action of thromboxane synthase product. These two enzymes have a typical cytochrome P450 heme center but catalyze atypical efficient peroxidase-like reaction instead of a monooxygenase reaction.

At the front of NO signaling, nitric oxide synthase (NOS), the key enzyme that catalyzes the conversion from L-arginine to NO is another P450-like heme-containing protein. The catalysis involves a complex interplay of three substrates, four different enzyme cofactors and prosthetic groups to achieve a 5-electron oxidation of the guanidine nitrogen in L-arginine. The redox characteristics of each enzyme cofactors will be carefully evaluated and the electron-transfer sequence will be systematically defined in isolated NOS domains and the intact NOS proteins. Recent focus is to characterize the reaction mechanisms of three NOS isozymes in the presence and absence of substrate L-arginine/N-hydroxyl-L-arginine, tetrahydrobiopterin and thiol to understand why NOS can behave as a superoxide synthase, hydrogen peroxide synthase or even peroxynitrite synthase at certain physiological and pathological conditions. We collaborate with Dr. Michael Bowman at University of Alabama, Birmingham to characterize the structures of various oxygen-induced radical intermediates under coupled or uncoupled conditions. The downstream enzyme that serves as the NO target, soluble guanylate cyclase (sGC), is another fascinating heme-containing protein to be investigated in collaboration with Drs. Emil Martin in my department, John S. Olson at Rice University and Focco van den Akker at Case Western Reserve. We will focus on elucidating the structural basis for massive activation of sGC activity by NO binding to the heme center and resolving the mechanism that underlies this activation process. To understand the ligand selectivity and the alternative mechanism in sensing redox potential changes, we also study other heme sensors that has  sequence and structure diverging from sGC.  We recently derived a "sliding scale rule" from bioinformatic analysis of binding parameters, KD, kon, and koff for NO, CO and O2 from a panel of hemeproteins that have been published or recently acquired by us. This new rule serves as our testing hypothesis and a guidance in design future heme-based sensors. We will collaborate with structural biologist, computation expert, biophysicist and molecular biologist to tackle this interesting ligand selectivity issue as well as the structure/function and reaction mechanism of sGC and other individual sensors.

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Selected References

Lü, J.-M., Rogge, R.E., Wu, G., Kulmacz, R.J., van der Donk, W.A. and Tsai, A.-L. Reaction mechanism of PGHS evidence for a reversible transition between a pentadienyl radical and a new tyrosyl radical by nitric oxide trapping. J. Inorg. Biochem. 105, 238-247, 2011.

Tsai, A.-L., Wu, G., Rogge, C.E., Lü, J.-M., Peng, S., van der Donk, W.A., Palmer, G., Gerfen, G.J. and Kulmacz, R.J. Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin h synthase-1 and -2. J. Inorg. Biochem. 105, 248-256, 2011.

Wu, G., Lü, J.-M., van der Donk, W.A., Kulmacz, R.J. and Tsai, A.-L. Cyclooxygenase reaction mechanism of prostaglandin H synthase from deuterium kinetic isotope effects. J. Inorg. Biochem. 105, 264-272, 2011.

Sasaki, J., Tsai, A.-L. and Spudich, J.L. Opposite displacement of helix F in attractant and repellent signaling by sensory rhodopsin-Htr complexes. J. Biol. Chem. 286, 18868-18877, 2011.

Liu, W., da Silva, G.F., Wu, G., Tsai, A.-L. and Kulmacz, R.J. Functional and structural roles of residues in the third extra-membrane segment (EM3) of adrenal cytochrome b561. Biochemistry, 50, 3149-3160, 2011.

Liu, W., Wu, G., Tsai, A.-L. and Kulmacz, R.J. High-yield production, purification and characterization of functional human duodenal cytochrome b in an Escherichia coli system. Protein Expr. Purif. 79, 115-121, 2011.

Wu, G., Berka, V., and Tsai, A.-L. Binding kinetics of calmodulin with target peptides of three nitric oxide synthase isozymes. J. Inorg. Biochem. 105, 1226-1237,  2011.

Tsai, A. -L., Berka, V., Sharina, I., and Martin, E. Dynamic ligand exchange in soluble guanylyl cyclase: implications for sGC regulation and desensitization. J. Biol. Chem. 286, 43182-43192, 2011.

Tsai, A. -L., Berka, V., Martin, E. and Olson, J. S. A "Sliding-Scale Rule" for Selectivity between NO, CO and O2 by Heme Protein Sensors. Biochemistry, in press.

Search PubMed for a complete list of Dr. Tsai's publications.