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1.
J Biol Chem ; 275(47): 36876-84, 2000 Nov 24.
Article in English | MEDLINE | ID: mdl-10973949

ABSTRACT

The serine protease domain of factor Xa (FXa) contains a sodium as well as a calcium-binding site. Here, we investigated the functional significance of these two cation-binding sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na(+) binds to the substrate bound FXa with K(d) approximately 39 mm in the absence and approximately 9.5 mm in the presence of Ca(2+). Sodium-bound FXa (sodium-Xa) has approximately 18-fold increased catalytic efficiency ( approximately 4.5-fold decrease in K(m) and approximately 4-fold increase in k(cat)) in hydrolyzing S-2222 (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide), and Ca(2+) further increases this k(cat) approximately 1.4-fold. Ca(2+) binds to the protease domain of substrate bound FXa with K(d) approximately 705 microm in the absence and approximately 175 microm in the presence of Na(+). Ca(2+) binding to the protease domain of FXa (Xa-calcium) has no effect on the K(m) but increases the k(cat) approximately 4-fold in hydrolyzing S-2222, and Na(+) further increases this k(cat) approximately 1.4-fold. In agreement with the K(m) data, sodium-Xa has approximately 5-fold increased affinity in its interaction with p-aminobenzamidine (S1 site probe) and approximately 4-fold increased rate in binding to the two-domain tissue factor pathway inhibitor; Ca(2+) (+/-Na(+)) has no effect on these interactions. Antithrombin binds to Xa-calcium with a approximately 4-fold faster rate, to sodium-Xa with a approximately 24-fold faster rate and to sodium-Xa-calcium with a approximately 28-fold faster rate. Thus, Ca(2+) and Na(+) together increase the catalytic efficiency of FXa approximately 28-fold. Na(+) enhances Ca(2+) binding, and Ca(2+) enhances Na(+) binding. Further, Na(+) enhances S1 site occupancy, and S1 site occupancy enhances Na(+) binding. Therefore, Na(+) site is thermodynamically linked to the S1 site as well as to the protease domain Ca(2+) site, whereas Ca(2+) site is only linked to the Na(+) site. The significance of these findings is that during physiologic coagulation, most of the FXa formed will exist as sodium-Xa-calcium, which has maximum biologic activity.


Subject(s)
Calcium/metabolism , Endopeptidases/metabolism , Factor Xa/metabolism , Sodium/metabolism , 4-Aminobenzoic Acid/metabolism , Binding Sites , Catalysis , Drug Synergism , Humans , Kinetics , Lipoproteins/metabolism , Models, Molecular , Oligopeptides/metabolism , Protein Binding , Protein Conformation , Protein Folding , Thermodynamics
3.
Chem Res Toxicol ; 5(1): 42-53, 1992.
Article in English | MEDLINE | ID: mdl-1581535

ABSTRACT

Testosterone (T) 6 beta-hydroxylase (6 beta-OHase) is a well-recognized functional marker of rat liver cytochrome P450 3A (P450 3A) isozymes. Pretreatment of rats with inducers or specific or nonspecific inhibitors of P450 3A isozymes is associated not only with stimulation or inhibition of hepatic microsomal T 6 beta-OHase activity but also with parallel changes in the corresponding T 2 beta-, 15 beta-, and 18-OHase activities and T 4,6-diene formation. At the time these studies were conducted, no fully functionally active rat hepatic P450 3A isozymes had been isolated. To determine whether each of these activities was due to a single P450 3A isozyme, or whether multiple isozymes contributed to these activities, we specifically synthesized two thiotestosterone (6 beta- and 2 beta-SHT) analogues as potential mechanism-based inactivators of rat liver T 6 beta- and 2 beta-OHases. In addition, to assess their relative stereoselectivity, 2 alpha-SHT was also included as a control. Our studies revealed that although all three thiosteroids were excellent suicide substrates of P450 3A isozymes, they inactivated these T OHases differentially. Such differential inactivation and determination of the kinetic parameters of inactivation allowed the functional classification of rat hepatic P450 3A isozymes into at least two and possibly three categories: (i) forms (catalyzing 4,6-diene and 6 beta-OHT formation but with characteristically low 6 beta/2 beta-OHase ratios) highly susceptible to inactivation by SHTs; (ii) forms (catalyzing T 6 beta-, 2 beta-, 15 beta-, and 18-hydroxylations with high 6 beta-/2 beta-OHase ratios) moderately susceptible to the SHTs; and (iii) forms somewhat resistant to inactivation, at least at the SHT concentrations tested. Although no specific T OHase could be ascribed to a single P450 3A isozyme, it appears that each of these P450s catalyzed such T regiohydroxylations, albeit at considerably different extents. Furthermore, our studies also revealed that 2 alpha-SHT preferentially inactivated P450 3A forms but surprisingly failed to inactivate rat hepatic P450 2C11, thereby confirming the rather high substrate promiscuity of the P450 3A family of isozymes.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Isoenzymes/metabolism , Liver/enzymology , Sulfhydryl Compounds/chemical synthesis , Testosterone/analogs & derivatives , Testosterone/chemical synthesis , Animals , Binding Sites , Cytochrome P-450 Enzyme Inhibitors , Dexamethasone/pharmacology , In Vitro Techniques , Kinetics , Magnetic Resonance Spectroscopy , Male , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Phenobarbital/pharmacology , Rats , Rats, Inbred Strains , Stereoisomerism , Steroid Hydroxylases/antagonists & inhibitors , Steroid Hydroxylases/metabolism , Testosterone/metabolism
4.
Mol Pharmacol ; 32(1): 299-308, 1987 Aug.
Article in English | MEDLINE | ID: mdl-3302670

ABSTRACT

In vivo administration of the porphyrogenic agent allylisopropylacetamide (AIA) to phenobarbital-pretreated rats results in marked loss of hepatic cytochrome P-450 content. Using isozyme-selective functional markers, we now show that such loss reflects inactivation of several phenobarbital-inducible and constitutive isozymes. Some of the isozymes (P-450a,b,h and PB-1) are largely reparable by reconstitution with exogenous hemin, indicating that after AIA-mediated loss of their prosthetic heme, their apoprotein moieties are essentially intact and functionally reconstitutable with hemin. On the other hand, after AIA-mediated inactivation, isozymes such as cytochrome P-450p remain refractory to such repair. The cause for such intractability remains somewhat elusive since AIA-mediated alkylation of the apocytochrome, proteolytic loss of the hemoprotein, or even irreversible binding of prosthetic heme catabolites to the apocytochrome does not appear to be responsible.


Subject(s)
Acetamides/administration & dosage , Allylisopropylacetamide/administration & dosage , Cytochrome P-450 Enzyme Inhibitors , Liver/enzymology , Alkylation , Allylisopropylacetamide/immunology , Animals , Biotransformation , Drug Administration Schedule , Hemin/administration & dosage , Immunologic Techniques , Isoenzymes/antagonists & inhibitors , Rats
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