Oxidation Of Inosine Research Articles

Simultaneous detection of ATP metabolites in human plasma and urine based on palladium nanoparticle and poly(bromocresol green) composite sensor

A sensitive voltammetric sensor based on palladium nanoparticles (PdNPs) and poly-bromocresol green (pBG) composite layer immobilized on amide functionalized single-walled carbon nanotubes (AmSWCNTs) modified pyrolytic graphite (PdNPs:pBG/AmSWCNTs/PG) has been prepared for the simultaneous determination of adenosine triphosphate (ATP) catabolites, inosine (INO), hypoxanthine (HX), xanthine (XT), and uric acid (UA). The modified PdNPs:pBG/AmSWCNTs/PG was characterized by electrochemical experiments and surface analysis, which exhibited exceptional electrocatalytic effects towards the oxidation of INO, HX, XT, and UA with a significant enhanced peak current and well resolved peaks separation for all the analytes. The linear calibration curves were obtained in the concentration range of 0.001–175 µM, 0.001–200 µM, 0.001–150 µM, and 0.001–200 µM and limits of detection were found as 0.95 nM, 1.04 nM, 1.07 nM, and 0.43 nM corresponding to INO, HX, XT, and UA, respectively. The common metabolites present in the biological fluids did not interfere in the determination. The applicability of the proposed sensor was successfully demonstrated by determining INO, HX, XT, and UA in the human plasma and urine and the obtained results were validated by using HPLC.

Electrochemical oxidation of inosine-5′-triphosphate at pyrolytic graphite electrode

The oxidation chemistry of inosine-5′-triphosphate (5′-ITP) has been investigated in phosphate containing supporting electrolytes of pH range 2.23–10.42 at pyrolytic graphite electrode (PGE). In cyclic voltammetry, a well-defined oxidation peak Ia was noticed over the entire pH range. The peak potential of peak Ia shifted to less positive potential when pH was increased. Concentration and sweep rate studies established the adsorption of 5′-ITP on the electrode surface. UV–vis spectral analysis indicated the formation of a UV-absorbing intermediates, generated during electrooxidation which decayed in a pseudo-first-order reaction having rate constant (k ∼ 0.672–0.783) × 10−3 s−1. The products were separated by HPLC and characterized by GC–MS, FT-IR, mp and 1H NMR as allantoin, C O O C, N N, C C bridged dimers, tetramer and ribose. A detailed interpretation of the redox mechanism of inosine-5′-triphosphate has been presented and a comparison of the observed electrochemical behavior with inosine indicated that Ep shifted to more positive potentials in the presence of phosphate groups. Many of the observed products were essentially similar to that observed during oxidation of inosine.

Is Arg418 the Catalytic Base Required for the Hydrolysis Step of the IMP Dehydrogenase Reaction?

The first committed step of guanine nucleotide biosynthesis is the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) catalyzed by IMP dehydrogenase. The reaction involves the reduction of NAD(+) with the formation of a covalent enzyme intermediate (E-XMP). Hydrolysis of E-XMP requires the enzyme to adopt a closed conformation and is rate-limiting. Thr321, Arg418, and Tyr419 are candidates for the residue that activates water. The substitution of Thr321 has similar, but small, effects on both the hydride transfer and hydrolysis steps. This result suggests that Thr321 influences the reactivity of Cys319, either through a direct interaction or by stabilizing the structure of the active site loop. The hydrolysis of E-XMP is accelerated by the deprotonation of a residue with a pK(a) of approximately 8. A similar deprotonation stabilizes the closed conformation; this residue has a pK(a) of >or=6 in the closed conformation. The substitution of Tyr419 with Phe does not change the pH dependence of either the hydrolysis of E-XMP or the conformational change, which suggests that Tyr419 is not the residue that activates water. In contrast, the conformational change becomes pH-independent when Arg418 is substituted with Gln. Lys can replace the function of Arg418 in the hydrolysis reaction but does not stabilize the closed conformation. The simplest explanation for these observations is that Arg418 serves as the base that activates water in the IMPDH reaction.

The Use of Inosine 5′-Monophosphate Dehydrogenase (IMPDH) in the Development of a New Liquid Homogeneous Enzyme Immunoassay Technology

The objective of the present study was the development of a quantitative liquid homogeneous immunoassay specific for theophylline based on the specific uncompetitive inhibition of inosine 5′-monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). This was accomplished by covalent coupling of theophylline to MPA to form a theophylline-MPA conjugate (Fig. 1⇓ ). IMPDH (EC 1.1.1.205) (1) catalyzes the NAD-dependent oxidation of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). The enzyme follows an ordered Bi-Bi reaction sequence of substrate and cofactor binding and product release. In the first step, IMP binds to IMPDH, followed by the binding of the cofactor NAD. After IMP oxidation, the reduced cofactor, NADH, is released from IMPDH, followed by the release of XMP. To monitor the reaction, the rate of NADH formation is measured at 340 nm. Uncompetitive inhibition occurs when MPA combines with the IMPDH-XMP complex at the active site of the enzyme to form IMPDH-XMP-MPA complex, which is unable to release XMP. IMPDH inhibition depends only on the concentration of MPA because of the uncompetitive nature of inhibition by MPA. Thus, the greater the concentration of MPA inhibitor, the greater the inhibition of the enzyme. An uncompetitive inhibitor of IMPDH inhibits by binding at the active site of the enzyme and does not compete with IMP …