Recombinant Escherichia coli GMP reductase: kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme–ligand binary complex formation

Abstract

Guanosine monophosphate (GMP) reductase catalyzes the reductive deamination of GMP to inosine monophosphate (IMP). GMP reductase plays an important role in the conversion of nucleoside and nucleotide derivatives of guanine to adenine nucleotides. In addition, as a member of the purine salvage pathway, it also participates in the reutilization of free intracellular bases. Here we present cloning, expression and purification of Escherichia coli guaC-encoded GMP reductase to determine its kinetic mechanism, as well as chemical and thermodynamic features of this reaction. Initial velocity studies and isothermal titration calorimetry demonstrated that GMP reductase follows an ordered bi-bi kinetic mechanism, in which GMP binds first to the enzyme followed by NADPH binding, and NADP(+) dissociates first followed by IMP release. The isothermal titration calorimetry also showed that GMP and IMP binding are thermodynamically favorable processes. The pH-rate profiles showed groups with apparent pK values of 6.6 and 9.6 involved in catalysis, and pK values of 7.1 and 8.6 important to GMP binding, and a pK value of 6.2 important for NADPH binding. Primary deuterium kinetic isotope effects demonstrated that hydride transfer contributes to the rate-limiting step, whereas solvent kinetic isotope effects arise from a single protonic site that plays a modest role in catalysis. Multiple isotope effects suggest that protonation and hydride transfer steps take place in the same transition state, lending support to a concerted mechanism. Pre-steady-state kinetic data suggest that product release does not contribute to the rate-limiting step of the reaction catalyzed by E. coli GMP reductase.

Helicobacter pylori Relies Primarily on the Purine Salvage Pathway for Purine Nucleotide Biosynthesis

“…These nucleotides can then be fed into the nucleotide biosynthesis pathway, where they can then be used to generate GMP or AMP. Additionally, based on the established pathway, enzymes such as GMP reductase (GuaC) and adenine/adenosine deaminases (Add/Ade) allow the cycling of nucleotides by creating bypasses in the pathway, thereby allowing for various purine bases to be used for generating both GMP and AMP, instead of only one or the other (46,55).…”

The dynamic determinants of reaction specificity in the IMPDH/GMPR family of (β/α)8barrel enzymes

“…The enzyme has been purified from a handful of sources: Aerobacter aerogenes (Mager and Magasanik, 1960, Brox and Hampton, 1968), human erythrocytes (Mackenzie and Sorensen, 1973, Spector, 1979 #838), calf thymus (Stephens and Whittaker, 1973), Leishmania donvani (Spector and Jones, 1982, Spector et al, 1984), Artemia salina (Renart et al, 1976a, Renart et al, 1976b), though its presence has been inferred in several other cases by the conversion of labeled guanine into adenine nucleotides. Only the human and Escherichia coli genes have been cloned and expressed to verify activity (Andrews and Guest, 1988, Moffat and Mackinnon, 1985, Martinelli et al, 2011, Patton et al, 2011, Li et al, 2006, Zhang et al, 2003, Deng et al, 2002). X-ray crystal structures are available for human GMPR1 and GMPR2 in several complexes: E•GMP (2ble, 2bwg, 2a7r, (Li et al, 2006)), E•IMP (2bzn, (Patton et al, 2011)) and E•IMP•NADH (2c6q, (Patton et al, 2011)).…”

“…This mechanism would be described by ping-pong kinetics, which are most definitely not observed in GMPRs. Instead, an intersecting line pattern is found in Lineweaver-Burk plots (Spector et al, 1979, Deng et al, 2002, Martinelli et al, 2011, Patton et al, 2011), indicating that a ternary E•GMP•NADPH complex must form before deamination can proceed. Further, again by analogy to the IMPDH reaction, the deamination reaction would be expected to be rate-limiting and hydride transfer is expected to be fast.…”

“…Further, again by analogy to the IMPDH reaction, the deamination reaction would be expected to be rate-limiting and hydride transfer is expected to be fast. However, two investigations of the mechanism of E. coli GMPR report that an isotope effect is observed on hydride transfer, indicating that in fact the deamination step is fast and hydride transfer is rate-limiting (Martinelli et al, 2011, Patton et al, 2011). These findings create a conundrum: how can the deamination reaction occur in the presence of cofactor?…”

“…However, an alternative concerted deamination/hydride transfer mechanism is also plausible, wherein Cys acts as a general acid to protonate the leaving group as hydride is transferred (Martinelli et al, 2011). Since hydride transfer is rate-limiting, if E-XMP* forms, it should accumulate in the steady state, and in fact the presence of E-XMP* has been demonstrated by mass spectroscopy (Patton et al, 2011).…”