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Titolo:
PRE-STEADY-STATE TRANSITION-STATE ANALYSIS OF THE HYDROLYTIC REACTIONCATALYZED BY PURINE NUCLEOSIDE PHOSPHORYLASE
Autore:
KLINE PC; SCHRAMM VL;
Indirizzi:
ALBERT EINSTEIN COLL MED,DEPT BIOCHEM,1300 MORRIS PK AVE BRONX NY 10461 ALBERT EINSTEIN COLL MED,DEPT BIOCHEM BRONX NY 10461
Titolo Testata:
Biochemistry
fascicolo: 4, volume: 34, anno: 1995,
pagine: 1153 - 1162
SICI:
0006-2960(1995)34:4<1153:PTAOTH>2.0.ZU;2-P
Fonte:
ISI
Lingua:
ENG
Soggetto:
AMP NUCLEOSIDASE; KINETIC MECHANISM; CALF-SPLEEN; INOSINE; ACID; INTERMEDIATE; INHIBITORS; HYDROLASE; SECONDARY; SUGARS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
43
Recensione:
Indirizzi per estratti:
Citazione:
P.C. Kline e V.L. Schramm, "PRE-STEADY-STATE TRANSITION-STATE ANALYSIS OF THE HYDROLYTIC REACTIONCATALYZED BY PURINE NUCLEOSIDE PHOSPHORYLASE", Biochemistry, 34(4), 1995, pp. 1153-1162

Abstract

The slow hydrolytic reaction catalyzed by calf spleen purine nucleoside phosphorylase [Kline, P. C., and Schramm, V. L. (1992) Biochemistry31, 5964-5973] has been investigated using pre-steady-state kinetic isotope effects and solvolysis studies. The stoichiometric reaction between enzyme and inosine forms 1 mol of free ribose per trimer of purine nucleoside phosphorylase and a tightly bound complex of enzyme and hypoxanthine. The experimental kinetic isotope effects from [1'-H-3]-, [2'-H-3]-, [4'-H-3]-, [5'-H-3]-, [1'-C-14]-, and [9-N-15]inosine are 1.151 +/- 0.004, 1.145 +/- 0.003, 1.006 +/- 0.004, 1.028 +/- 0.005, 1.045 +/- 0.005, and 1.000 +/- 0.005, respectively, for the pre-steady-state conditions. Substrate trapping experiments demonstrated that thereis no detectable forward commitment to catalysis for inosine hydrolysis. In contrast, bound inosine is 2.1 times more likely to form product than to dissociate when the enzyme-inosine complex is exposed to saturating PO4. The lack of an observed 9-N-15 isotope effect is consistent with an internal equilibrium between enzyme-inosine and the enzyme-hypoxanthine-ribose complex in which N9 of hypoxanthine is protonated. The equilibrium occurs as a consequence of slow product release and tightly bound hypoxanthine (K-d = 1.3 x 10(-12) M). This internal equilibrium has a minimal effect on the intrinsic kinetic isotope effects from ribose since equilibrium isotope effects for conversion of inosineto ribose are near unity. When the single-turnover hydrolytic reaction was accomplished in 20% methanol, approximately 85% of the product sugar was 1-methylribose. Under these conditions, the anion-binding pocket fills with solvent which competes for the oxocarbenium ion of inosine formed at the transition state. In the presence of arsenate, no methanolysis of inosine occurs [Kline, P. C., and Schramm, V. L. (1993) Biochemistry 32, 13212-13219]. The results define a transition state with oxocarbenium ion character and weak participation of the attackingsolvent nucleophile. Electrostatic potential surfaces of the transition states indicate that arsenate anion is more effective in neutralizing the oxocarbenium ion than is H2O.

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Documento generato il 18/09/20 alle ore 09:51:27