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. 2000 Oct 18;122(41):9911-9916.
doi: 10.1021/ja0021058.

Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions

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Free PMC article

Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions

June M Messmore et al. J Am Chem Soc. .
Free PMC article

Abstract

Pentavalent organo-vanadates have been put forth as transition state analogues for a variety of phosphoryl transfer reactions. In particular, uridine 2',3'-cyclic vanadate (U>v) has been proposed to resemble the transition state during catalysis by ribonuclease A (RNase A). Here, this hypothesis is tested. Lys41 of RNase A is known to donate a hydrogen bond to a nonbridging phosphoryl oxygen in the transition state during catalysis. Site-directed mutagenesis and semisynthesis were used to create enzymes with natural and nonnatural amino acid residues at position 41. These variants differ by 10(5)-fold in their k(cat)/K(m) values for catalysis, but <40-fold in their K(i) values for inhibition of catalysis by U>v. Plots of logK(i) vs log(K(m)/k(cat)) for three distinct substrates [poly(cytidylic acid), uridine 3'-(p-nitrophenyl phosphate), and cytidine 2',3'-cyclic phosphate] have slopes that range from 0.25 and 0.36. These plots would have a slope of unity if U>v were a perfect transition state analogue. Values of K(i) for U>v correlate weakly with the equilibrium dissociation constant for the enzymic complexes with substrate or product, indicating that U>v bears some resemblance to the substrate and product as well as the transition state. Thus, U>v is a transition state analogue for RNase A, but only a marginal one. This finding indicates that a pentavalent organo-vanadate cannot necessarily be the basis for a rigorous analysis of the transition state for a phosphoryl transfer reaction.

Figures

Figure 1
Figure 1
(A) Putative mechanism of the transphosphorylation reaction (top) and hydrolysis reaction (bottom) catalyzed by ribonuclease A. “B” is His12, and “A” is His119. (B) Putative structure of the transition state during the transphosphorylation reaction (HOR = nucleoside) or hydrolysis reaction (HOR = H2O) catalyzed by RNase A. (C) Stereoview of the structure of the active site in the RNase A•U>v complex. The structure was refined to 2.0 Å from X-ray and neutron diffraction data collected from crystals grown at pH 5.3 (Protein Data Bank entry 6RSA). The distance from Lys41 to the 2′ oxygen is 2.8 Å, and to the nearest nonbridging oxygen is 3.5 Å. The distance from His12 (right) to the nearest oxygen is 2.7 Å, and to the 2′ oxygen is 3.0 Å. The other enzymic residue is His119.
Figure 2
Figure 2
Plot of log(Ki/[U>v]) vs log(Km/kcat) for (A) cleavage of poly(cytidylic acid), (B) cleavage of Up(OC6H4-p-NO2), and (C) hydrolysis of cytidine 2′,3′-cyclic phosphate by wild-type ribonuclease A and the K41CEA, K41R, and K41A variants. Values of kcat/Km were obtained at 25 °C in 0.10 M MES-NaOH buffer (pH 6.0) containing NaCl (0.10 M). Values of Ki/[U>v] (Table 1) were obtained at 25 °C in 0.010 M sodium succinate buffer (pH 6.0) containing NaCl (0.10 M) and poly(C). Open circles (○) denote 15 mM uridine and 0.1 mM NaVO3 and have (A) slope = 0.25 ± 0.12, (B) slope = 0.31 ± 0.15, and (C) slope = 0.34 ± 0.12. Closed circles (●) denote 15 mM uridine and 0.4 mM NaVO3 and have (A) slope = 0.29 ± 0.16, (B) slope = 0.36 ± 0.18, and (C) slope = 0.36 ± 0.15. The dashed lines have a slope of unity.
Figure 3
Figure 3
Plot of log(Ki/[U>v]) vs logKm and logKp for wild-type ribonuclease A and the K41CEA, K41R, and K41A variants. Values of Ki/[U>v] (Table 1) were obtained at 25 °C in 0.010 M sodium succinate buffer, (pH 6.0) containing NaCl (0.10 M) and Up(OC6H4-p-NO2). Values of Km (●) were obtained from initial velocity data at 25 °C in 0.025 M MES-NaOH buffer (pH 6.0) containing NaCl (0.10 M) and C>p. Values of Kp (○; Table 2) were obtained from initial velocity data measured at 25 °C in 0.10 M MES-NaOH buffer, pH 6.0, containing NaCl (0.10 M), Up(OC6H4-p-NO2), and 3′-UMP. Values of Kp (□; Table 2) for the wild-type and K41A enzymes were also obtained by isothermal titration calorimetry of enzyme•3′-UMP complex formation. The solid line has slope ≈ 1.5; the dashed line has a slope of unity.
Figure 4
Figure 4
Plot of logKp vs log(kcat/Km) for wild-type ribonuclease A and the K41CEA, K41R, and K41A variants. Values of Kp (○; Table 2) were obtained from initial velocity data measured at 25 °C in 0.10 M MES-NaOH buffer, pH 6.0, containing NaCl (0.10 M), Up(OC6H4-p-NO2), and 3′-UMP. Values of kcat/Km were obtained at 25 °C in 0.025 M MES-NaOH buffer (pH 6.0) containing NaCl (0.10 M) and C>p. Values of Kp (□; Table 2) for the wild-type and K41A enzymes were also obtained by isothermal titration calorimetry of enzyme•3′-UMP complex formation. The solid line has slope ≈ 0.2; the dashed line has a slope of unity.
Figure 5
Figure 5
Free energy profile of catalysis by ribonuclease A and its inhibition by uridine 2′,3′-cyclic vanadate. The left profile depicts the free energy barrier for the uncatalyzed hydrolysis of C>p [ΔGuncat = −RTln(kuncath/kbT) ref 28] and the free energy barriers for hydrolysis of C>p catalyzed by the wild-type enzyme and three variants [ΔGtx° = − RTln(kcat/Km)/kuncat]. The right profile depicts the binding of these variants to the uridine vanadate complex [ΔGi° = −RTln(M/Ki)]. For wild-type RNase A, Ki = 0.45 μM. For the variant enzymes, Ki is calculated from the values of Ki/[U>v] (Table 1). To ease comparisons, the free energy of the inhibitor ‘I’ in the right profile is set equal to that of the transition state ,S, in the left profile.

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