Ah-Lim Tsai, Ph.D., Professor
The University of Texas Health Science Center at Houston
Medical School Department of Internal Medicine
Division of Hematology
6431 Fannin, MSB 5.290
Houston, Texas 77030

 

 

Tel: 713-500-6771
Fax: 713-500-6810
E-mail: Ah-Lim.Tsai@uth.tmc.edu

Ongoing Research Topics

The central theme of my research is to understand the reaction mechanism of several crucial heme-containing proteins that are involved in regulatory functions and the pathological processes of the cardiovascular system. Prostaglandins and nitric oxide (NO) are two important mediators in hemostasis. For example, prostacyclin and NO released by endothelial cells are strong vasodilators and potent inhibitors of platelet aggregation, whereas thromboxane and prostaglandin G2/H2 released by platelets are potent agonists of platelet aggregation and vasoconstriction. All these compounds are short-lived autocoids. A balanced production and timely release of these potent hormones are crucial in maintaining a normal vascular tone and an imbalanced synthesis of these mediators usually leads to pathological conditions.

The key enzymes responsible for the biosynthesis of prostaglandins and NO are prostaglandin H synthase (PGHS) and nitric oxide synthase (NOS), respectively. Both enzymes are hemeproteins and their catalysis involves complicated redox reactions. Co-localization of NOS and PGHS in different cells and crosstalk between the prostaglandin and NO biosynthesis have been reported extensively in the last few years. Crosstalk could occur at the protein or the gene level. A thorough understanding of the reaction mechanism of each enzyme will be very helpful in elucidating the underlying mechanism of the crosstalk phenomena which occurs at the protein level.

PGHS, the target of aspirin and other nonsteroidal anti-inflammatory agents (NSAIDs), has two isozymes. Type I enzyme is constitutive and is responsible for housekeeping purposes, whereas the Type II enzyme is inducible in the presence of various growth factors or cytokines and is involved in pathological events. Developing selective inhibitors for the Type II enzyme has great impact in pharmacology and human health. However, it is critical to understand the detailed reaction mechanism in order to put this enzyme under full control. Our approach to this goal is to employ various spectroscopic methods and rapid kinetic measurements to characterize the reaction intermediates, to locate the rate-limiting steps and to define the rate constants of each step of catalytic reaction. Computer simulation or fitting is then performed to test the mechanistic model derived from the data. Iteration between the actual experiments and
computer modeling will eventually lead us to a converged mechanism which most properly interpret all the existing data.

A branched-chain radical mechanism shown below serves as our testing model which has gained substantial experimental supports including X-ray crystallographic data from both isozymes. In this scheme, PGHS shows two enzyme activities, a peroxidase which converts peroxides into alcohol and a cyclooxygenase which transforms arachidonic acid to a fatty acid peroxide, Prostaglandin G2. Initiation of the cyclooxygenase cycle requires the presence of peroxide and the key factor which links the two catalytic cycles is a tyrosyl radical. This tyrosyl radical acts as the immediate oxidant for arachidonic acid. The first unique feature of this enzyme is its nature of self-propagation. As long as sufficient supply of arachidonic acid and oxygen are present, a very small quantity of peroxide will trigger an explosive production of the potent prostaglandins. Such exponential release of potent hormone could be lethal and is fortunately moderated by a self-inactivation process. Each PGHS molecule has a limited lifetime and is destroyed by certain reaction intermediate(s) generated during catalysis. Our mission is to clarify the complicated processes involved in both the self-activation and the self-inactivation of PGHS.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to NO and L-citrulline. This hemeprotein turned out to be a cytochrome P450 rather than a standard b-type heme with a proximal histidine ligand as in myoglobin and PGHS. Specific to NOS and not the other P450s is that NOS being a self-sufficient P450 with a very polar substrate, L-arginine. In other words, NOS has a fully functioning P450 reductase domain containing FAD, FMN and a binding site for NADPH, tethered to an oxygenase domain which contains the binding sites for heme, oxygen and tetrahydra-biopterin (BH4). Calmodulin and calcium are needed for electron transfer from the reductase domain to the oxygenase domain. Like PGHS, NOS also comes with three different isozymes: neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). Both nNOS and eNOS are constitutive like Type I PGHS and iNOS is inducible by many different stimulators just as Type II PGHS. The main difference among these three isoforms is the tight-association of calmodulin to iNOS to keep this isoform constantly activated. This nature makes iNOS very effective in killing bacteria engulfed by the macrophage cells. Similar to the case of PGHS, developing inhibitors selective to each isozyme of NOS is important to human health. We decided to study both the structure-function relationship and the reaction mechanism to delineate those dynamic events which are not likely be resolved by X-ray or NMR analysis. Series of spectroscopic methods in combination with site-directed mutagenesis is used to evaluate the structure-function relationship to unravel the environment of heme, flavin and BH4 binding sites and the role of calmodulin in regulation of the relative position between heme and flavins.

The reaction mechanism of NOS is at least as complex as PGHS because there are three (heme, FAD, FMN) or perhaps four (including BH4) redox centers, three different substrates (NADPH, L-arginine and oxygen) involved in the catalysis and is compounded further with the regulation of activity by calmodulin and calcium. We will use similar methods applied to PGHS studies to investigate intact NOS and its individual domains to characterize the important intermediates and define the reaction rates of key steps in both the reductase domain and the oxygenase domain. The role of calmodulin/calcium and BH4 will be evaluated in the context of electron-transfer. Our immediate focus is on the eNOS but comparative mechanistic studies will be conduct in the future for all three different isozymes.

Recent Publications:
1. Tsai, A-L., Hsi, L.C., Kulmacz, R.J., Palmer, G. and Smith, W.L.: Characterization of the tyrosyl radicals in ovine prostaglandin H synthase-1 by isotope replacement and site-directed mutagenesis. J. Biol. Chem., 269:5085-5091, 1994.
2. Kulmacz, R.J., Palmer, G. and Tsai, A-L.: Reaction and free radical kinetics of prostaglandin H synthase with Mn protoporphyrin IX as prosthetic group. Biochemistry, 33, 5428-5438, 1994.
3. Tsai, A.-L : How does NO activate hemeproteins? FEBS Lett. 341, 141-145, 1994
4. Tsai, A.-L., Wei, C. and Kulmacz, R. J. Interaction between nitric oxide and prostaglandin H synthase. Arch. Biochem. Biophys. 313, 367-372, 1994.
5. Sanduja, S. K., Tsai, A.-L., Matijevic-Aleksic, N. and Wu, K. K. Kinetics of prostacyclin synthesis and prostaglandin H synthase (PGHS-1) turnover in a PGHS-1 overexpressed endothelial cell. Amer. J. Physiol. 267, c1459-c1466, 1994.
6. Chen, P.-F., Tsai, A.-L. and Wu, K. K. Cystein 184 of endothelial nitric oxide synthase is involved in heme coordination and catalytic activity. J. Biol. Chem. 269, 25062-25066, 1994.
7. Tsai, A.-L., Kulmacz, R. J. and Palmer, G. Spectroscopic evidence for reaction of prostaglandin H synthase-1 tyrosyl radical with arachidonic acid. J. Biol. Chem. 270, 10503-10508, 1995.
8. Wei, C., Kulmacz, R. J. and Tsai, A.-L. Comparison of branched-chain and tightly coupled reaction mechanisms for prostaglandin H synthase. Biochemistry, 34, 8499- 8512, 1995.
9. Chen, P.-F., Tsai, A.-L. and Wu, K. K. Cysteine 99 of endothelial nitric oxide synthase (NOS-III) is critical for tetrahydrobiopterin-dependent NOS-III stability and activity. Biochem. Biophys. Res. Commun. 215, 1119-1129, 1995.
10. Chen, P.-F., Tsai, A.-L. Berka, V. and Wu, K. K. Endothelial nitric oxide synthase:evidence for bidomain structure and successful reconstitution of catalytic activity from two separate domains generated by a baculovirus expression system. J. Biol. Chem. 271, 14631-14635, 1996.
11. Berka, V., Chen, P.-F. and Tsai, A.-L. Spatial relationship between L-arginine and heme binding sites of endothelial nitric oxide synthase. J. Biol. Chem.
271, 33293- 33300, 1996.
12. Tsai, A.-L., Berka, V., Chen, P,-F. and Palmer, G. Characterization of endothelial nitric oxide synthase and its reaction with ligand by electron
pramagnetic resonance spectroscopy. J. Biol. Chem. 271, 32563-32571, 1996.
13. Xiao, G., Tsai, A.-L., Palmer, G., Boyar, W.C., Marshall, P.J. and Kulmacz, R.J. Analysis of hydroperoxide-induced tyrosyl radicals and lipoxygenase activity in aspirin-treated human prostaglandin H synthase-2. Biochemistry, 36, 1836-1845, 1997.
14. Chen, P-F., Tsai, A.-L., Berka, V. and Wu, K.K. Mutation of glu-361 in human endothelial nitric-oxide synthase selectively abolishes L-arginine binding without perturbing the behavior of heme and other redox centers. J. Biol. Chem. 272, 6114- 6118, 1997.
15. Tsai, A.-L., Wei, C., Baek, H.K., Kulmacz, R.J. and Van Wart, H.E. Comparison of peroxidase reaction mechanisms of prostaglandin H synthase-1 containing heme and mangano protoporphyrin IX. J. Biol. Chem. 272, 8885-8894, 1997.
16. Tsai, A.-L., Wu, G. and Kulmacz, R.J. Stoichiometry of the interaction of prostaglandin H synthase with substrates. Biochemistry, 36, 13085-13094, 1997.
17. Tsai, A.-L., Palmer, G., Xiao, G., Swinney, D.C., and Kulmacz, R.J. Structural characterization of arachidonyl radicals formed by prostaglandin H synthase-2 and prostaglandin H synthase-1 reconstituted with mangano protoporphyrin IX. J. Biol. Chem. 273, 3888-3894. 1998.
18. Berka, V., Palmer, G., Chen, P-F., and Tsai, A-L. Effects of various imidazole ligands on heme conformation in endothelial nitric oxide synthase. Biochemistry, 37, 6136-6144, 1998.

Expertise:
Metelloenzymes, esp. Hemeproteins Electron transfer and redox reactions Enzyme kinetics, mainly transient kinetics (stopped-flow, rapid-freezing and rapid quench) Computer modeling and simulation Bioenergetics Mitochondria (structure and function) Membrane-bound proteins Prostaglandins, receptors, binding proteins and signal transduction Prostaglandin H synthase and nonsteroidal anti-inflammatory agents Nitric oxide and nitric oxide synthase Spectroscopic methods (UV-VIS, fluorescence, EPR and MCD).