Experimental charge measurement at leaving oxygen in the bovine ribonuclease. A catalyzed cyclization of uridine 3'-phosphate aryl esters

Abstract

The title esters are demonstrated to be specific substrates of bovine pancreatic ribonuclease A (EC 3.1.27.5). The Brønsted dependence of kcat/Km at pH 7.50 for the enzyme-catalyzed cyclization versus the pKa of the leaving phenol exhibits two regression lines of almost identical slope for respectively 2-chlorophenols and 2,6-unsubstituted phenols: log kcat/Km = -0.20 pKa ArOH + 5.47 (n = 5, r = 0.957); log kcat/Km = -0.17 pKa ArOH + 5.79 (n = 4, r = 0.965). Comparison of the Brønsted beta 1g's with that for the standard reaction where imidazole catalyzes the cyclization (beta 1g = -0.59) indicates considerably less development of negative charge on the leaving oxygen in the enzyme case, providing experimental evidence for the hypothesis that electrophilic assistance is involved in catalysis. The existence of two essentially parallel Brønsted correlations is not reflected in the standard reaction of substrate with imidazole. Modeling studies indicate that the phenyl ring of the substrate can take up a range of positions away from the active site; the presence of ortho chloro substituents considerably restricts the motion of the phenyl leaving group.

Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage

“…Consistent with this idea, a ␤ lg of Ϫ0.54 to Ϫ0.59 was determined for an aryl phosphate diester that bears a good leaving group (32). It has been shown that the ␤ lg for a series of aryl-phosphate diester compounds is even less negative (␤ lg ϭ Ϫ0.2) in the active site of RNase A, suggesting a considerable contribution of electrophilic catalysis in the active site (33). Brønsted analysis using aryl-phosphate diester compounds gave a Brønsted coefficient, ␤, of 0.67 (␣ of 0.33) (32,34).…”

Kinetic Isotope Effects and Stereochemical Studies on a Ribonuclease Model: Hydrolysis Reactions of Uridine 3′-Nitrophenyl Phosphate

“…This compound was reacted with remote labeled p-nitrophenyl phosphorodichloridate and purified as described above for the natural abundance compound. The compound was SCHEME 2 stored as the 2Ј,5Ј-bis(4-methoxytetrahydropyran-4-yl) ether and deblocked in dilute HCl solution just before use in measurements of bridge- 18 O isotope effects. The achiral 4-methoxytetrahydropyran-4-yl ether protecting group was used to avoid differential rates in the acid-catalyzed removal of the protecting groups which could occur with the chiral tetrahydropyranyl ether.…”

“…NMR experiments monitoring the removal of the tetrahydropyranyl groups versus hydrolysis showed that under these conditions deprotection was complete while hydrolysis of the deprotected diester was undetectable by 13 P NMR. For 15 N isotope effects, the natural abundance 2Ј,5Ј-bis(tetrahydropyranyl)uridine-3Ј-p-nitrophenyl phosphate (1a) was used, and for the 18 O isotope effect experiments, remote labeled 2Ј,5Ј-bis(4-methoxytetrahydropyran-4-yl) uridine-3Ј-p-nitrophenyl phosphate (1b) was used. It was noted that the four diastereomers of 1a resulting from the chiral tetrahydropyranyl groups exhibited slightly different rates of deprotection.…”

“…The reaction was complete within 3 h, as evidenced by the appearance of two groups of signals at 19.5 and 18.8 ppm. In addition to the two groups of signals arising from exo-and endomethyl esters of [ 16 O, 18 O] cUMP, the formation of a minor quantity of upfield (carbonate sample) and down field (imidazole sample) was observed ( Fig. 2).…”

“…The eluate was concentrated, passed through a Dowex 50-X8 column in the potassium form, and concentrated to give cUMP as its potassium salt. 18 O] cUMP (half of the total quantity) was transferred to a 5-mm NMR tube and freeze-dried. The solid residue was made anhydrous by repeated dispersion in warm, dry dioxane and freeze-drying.…”