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Titolo:
PURINE NUCLEOSIDE PHOSPHORYLASE - CATALYTIC MECHANISM AND TRANSITION-STATE ANALYSIS OF THE ARSENOLYSIS REACTION
Autore:
KLINE PC; SCHRAMM VL;
Indirizzi:
YESHIVA UNIV ALBERT EINSTEIN COLL MED,DEPT BIOCHEM,1300 MORRIS PK AVEBRONX NY 10461 YESHIVA UNIV ALBERT EINSTEIN COLL MED,DEPT BIOCHEM,1300 MORRIS PK AVEBRONX NY 10461
Titolo Testata:
Biochemistry
fascicolo: 48, volume: 32, anno: 1993,
pagine: 13212 - 13219
SICI:
0006-2960(1993)32:48<13212:PNP-CM>2.0.ZU;2-1
Fonte:
ISI
Lingua:
ENG
Soggetto:
AMP NUCLEOSIDASE; HYDROLYSIS; SECONDARY; INOSINE; ADENOSINE; BINDING; RIBOSE; ENZYME; ACID;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
46
Recensione:
Indirizzi per estratti:
Citazione:
P.C. Kline e V.L. Schramm, "PURINE NUCLEOSIDE PHOSPHORYLASE - CATALYTIC MECHANISM AND TRANSITION-STATE ANALYSIS OF THE ARSENOLYSIS REACTION", Biochemistry, 32(48), 1993, pp. 13212-13219

Abstract

Purine nucleoside phosphorylase from calf spleen catalyzes the arsenolysis of inosine to form hypoxanthine and ribose 1-arsenate, which spontaneously hydrolyzes to ribose and arsenate. In the presence of (H2O)-O-18, no O-18 is incorporated into ribose, demonstrating that ribose I-arsenate hydrolysis occurs by attack of water on the arsenic atom. Rapid reaction kinetics at 20-degrees-C result in a biphasic rate curvewith the first turnover occurring at a rate of 20 s-1 followed by a steady-state rate of 2 s-1. The product burst is consistent with rapid steps for substrate binding and arsenolysis followed by rate-limiting hypoxanthine release at a rate of 2 s-1. Purine nucleoside phosphorylase with bound [C-14]inosine was mixed with excess unlabeled inosine and arsenate to determine relative rates for reaction or dissociation ofbound inosine. The commitment factor (product formed/inosine released) was 0.19 at saturating arsenate, indicating that inosine binds to free enzyme and that bound inosine is not in thermodynamic equilibrium with free substrate. At neutral pH, kinetic isotope effects for the phosphorolysis reaction are small, indicating kinetic suppression. Kinetic isotope effects for arsenolysis were measured with [1'-H-3]-, [2'-H-3]-, [1'-C-14]-, [9-N-15]-, [4'-H-3]-, and [5'-H-3] inosine to provideexperimental values of 1.118 +/- 0.003, 1.128 +/- 0.003, 1.022 +/- 0.005, 1.009 +/- 0.004, 1.007 +/- 0.003 and 1.028 +/- 0.004 respectively. Following correction for commitment factors, the intrinsic isotope effects were matched to a geometric transition-state model selected by bond-energy bond order vibrational analysis. The transition state consistent with all isotope effects has a substantial decrease in the C1'-N9 glycosyl bond order, oxycarbonium character in the ribosyl ring, and weak participation of the arsenate nucleophile. Loss of the C1'-N9 bond is far ahead of the arsenate attack. The X-ray crystal structure for purine nucleoside phosphorylase with bound 9-deazainosine and inorganic sulfate places the nearest oxygen of the sulfate 4.2 angstrom from C1' of the nucleoside analogue. This structure is consistent with a mechanism in which the ribosyl group is nearly dissociated from the base prior to attack of the arsenate.

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Documento generato il 18/09/20 alle ore 11:02:54