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Titolo:
INHIBITOR-INDUCED CHANGES IN THE INTRINSIC FLUORESCENCE OF HUMAN CYCLOOXYGENASE-2
Autore:
HOUTZAGER V; OUELLET M; FALGUEYRET JP; PASSMORE LA; BAYLY C; PERCIVAL MD;
Indirizzi:
MERCK FROSST CTR THERAPEUT RES,DEPT BIOCHEM & MOL BIOL,POB 1005 POINTE CLAIRE PQ H9R 4P8 CANADA MERCK FROSST CTR THERAPEUT RES,DEPT BIOCHEM & MOL BIOL POINTE CLAIRE PQ H9R 4P8 CANADA MERCK FROSST CTR THERAPEUT RES,DEPT MED CHEM POINTE CLAIRE PQ H9R 4P8CANADA
Titolo Testata:
Biochemistry
fascicolo: 33, volume: 35, anno: 1996,
pagine: 10974 - 10984
SICI:
0006-2960(1996)35:33<10974:ICITIF>2.0.ZU;2-P
Fonte:
ISI
Lingua:
ENG
Soggetto:
PROSTAGLANDIN-H-SYNTHASE; NONSTEROIDAL ANTIINFLAMMATORY DRUGS; SELECTIVE-INHIBITION; ENDOPEROXIDE SYNTHASE-2; G/H SYNTHASE-1; ACTIVE-SITE; PROTEIN; ASPIRIN; ISOFORMS; PURIFICATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
41
Recensione:
Indirizzi per estratti:
Citazione:
V. Houtzager et al., "INHIBITOR-INDUCED CHANGES IN THE INTRINSIC FLUORESCENCE OF HUMAN CYCLOOXYGENASE-2", Biochemistry, 35(33), 1996, pp. 10974-10984

Abstract

The steady state tryptophan fluorescence of ape-human cyclooxygenase-2 (hCox-2) is quenched approximately 40%-50% by the slow binding inhibitors diclofenac, indomethacin, ketoprofen, NS-398, and DuP-697. The effects of these inhibitors on tryptophan fluorescence are both time and concentration dependent. Addition of each inhibitor results in a rapid fluorescence decrease, followed by a slower time dependent quenching. The slow, time dependent loss of fluorescence follows first-order kinetics, the rate constants for the process increasing with inhibitor concentration in a saturation-type manner, The rapid fluorescence lossalso increases with increasing inhibitor concentration in the same manner. These results an consistent with the initial formation of a rapid equilibrium complex of enzyme and inhibitor (EI), followed by the slower formation of a tightly bound enzyme-inhibitor complex (EI). The fluorescence of the EI complex is not significantly different from that of the EI complex. The kinetic parameters of each inhibitor derivedfor this process (K-i and k(on)) are close to those obtained by determination of the rate constants for the onset of enzyme inhibition, thereby linking the fluorescence changes with inhibitor binding. The reversible inhibitors ibuprofen and docosahexaenoic acid do not quench theprotein fluorescence but do decrease both the rate of the slow fluorescence loss and the magnitude of the initial rapid fluorescence decrease caused by the slow binding inhibitors, consistent with their competitive behavior. ASA-acetylated apo-hCox-2 shows the same fluorescence-quenching behavior in the presence of most of the above inhibitors. However, acetylation apparently blocks the binding of diclofenac, whereas the affinity of ibuprofen is increased. The effects of the collisional quenching agents iodide and acrylamide on both the native and inhibited enzyme are small (<20% quenching at 0.3 M), Showing that inhibitor binding does not result in an increased solvent accessibility of protein tryptophans. The cause of the inhibitor-induced quenching of the intrinsic apo-hCox-2 fluorescence is likely energy transfer to the bound inhibitor, Calculations based on the inhibitor-tryptophan distancesin ovine Cox-1 indicate that the distances are within the required range for significant quenching to occur.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 26/09/20 alle ore 05:46:13