Pyrophosphate activation in hypoxanthine--guanine phosphoribosyltransferase with transition state analogue

Abstract

Isotope-edited difference Raman and FTIR studies complemented by ab initio calculations have been applied to the transition state analogue complex of HGPRT.ImmHP.MgPP(i) to determine the ionic states of the 5'-phosphate moiety of ImmHP and of PP(i). These measurements characterize electrostatic interactions within the enzyme active site as deduced from frequency shifts of the phosphate groups. The bound 5'-phosphate moiety of ImmHP is dianionic, and this phosphate group exists in two different conformations within the protein complex. In one conformation, a hydrogen bond between the 5'-phosphate of ImmHP and the OH group of Tyr104 in the catalytic loop appears to be stronger. With the stronger H-bond, the OH of Tyr104 approaches one of the P..O bonds from the bridging oxygen side to cause distortion of the PO(3) moiety, as indicated by a lowered symmetric P..O stretch frequency. The asymmetric stretch frequencies are similar in both phosphate conformations. Bound PP(i) in this complex is fully ionized to P(2)O(7)(4-). Bond frequency changes for bound PP(i) indicate coordination to Mg(2+) ions but show no indication of significant P..O bond polarization. Extrapolation of these results to reaction coordinate motion for HGPRT suggests that bond formation between C1' of the nucleotide ribose and the oxygen of PP(i) is accomplished by migration of the ribocation toward immobilized pyrophosphate.

Figure 1

(a) Raman spectrum of 100 mM dianionic G6P at pH 8.5. (b) Raman spectrum of 100mM monoanionic G6P at pH 4.0. (c) Raman difference spectrum between HGPRT●ImmHP●MgPPi and HGPRT● ¹⁸O-ImmHP●MgPPi complex, at a enzyme concentration of 2mM. The enzyme samples were prepared in 10 mM tris at pH 7.5 with 5 mM DTT. The 514.5 nm line from an argon ion laser or 568.2 nm line from Krypton ion laser was used to irradiate the sample (∼100 mW). The spectral resolution was 8 cm⁻¹.

Figure 2

(a) FTIR spectrum of 100 mM G6P at pH 8.5. (b) FTIR spectrum of 100mM G6P at pH 4.0. (c) FTIR difference spectrum between HGPRT●ImmHP●MgPPi complex and HGPRT● ¹⁸O-ImmHP●MgPPi complex. The enzyme samples were the same as described in Figure 1. The spectral resolution was 2 cm⁻¹.

Figure 3

(a) Raman spectrum of 100 mM tetraanionic PPi (pH11). (b) Raman spectrum of trianionic PPi. This spectrum is obtained by subtracting appropriate amount of a and c from the spectrum of 100 mM PPi at pH7.5. (c) Raman spectrum of 100mM dianionic PPi (pH 4.5). (d) Raman difference spectrum between HGPRT●ImmHP●MgPPi complex and HGPRT●ImmHP●Mg ¹⁸O-PPi complex. The experimental conditions were the same as described in Figure 1.

Figure 4

(a) FTIR spectrum of 100 mM tetraanionic PPi (pH11). (b) FTIR spectrum of 100 mM PPi. This spectrum is obtained by subtracting appropriate amount of a from the spectrum of 100 mM PPi at pH8.0. (c) FTIR spectrum of 100mM dianionic PPi (pH 4.5). (d) FTIR difference spectrum of HGPRT●ImmHP●MgPPi complex and HGPRT●ImmHP●Mg ¹⁸O-PPi complex. The experimental conditions were the same as described in Figure 2.

Scheme 1

Reaction catalyzed by HGPRT and its putative transition state.

Scheme 2

Active site contacts in the HGPRT/immucillinGP/PPi complex (taken from ref. 16).

Scheme 3

Model PPi compounds used in vibrational normal mode calculations.