CHARACTERIZATION OF H-PARASUIS PERIPLASMIC NUCLEOTIDE PYROPHOSPHATASEAS A POTENTIAL TARGET ENZYME FOR INHIBITION OF GROWTH

The periplasmic nucleotide pyrophosphatase from Haemophilus parasuis w as purified 750-fold to electrophoretic homogeneity through salt fract ionation and ion-exchange and affinity chromatography. The purified en zyme was monomeric with an apparent M-r, of 70,000 and catalyzed the h ydrolysis of the pyrophosphate bond of NAD to yield NMN and AMP as pro ducts. The enzyme exhibited negative cooperativity in the hydrolysis o f a number of pyridine dinucleotides and structurally-related pyrophos phate compounds as indicated by biphasic double-reciprocal plots and H ill coefficients of 0.5. The kinetic parameters, K-m, end V-m, determi ned titrimetrically and analyzed through computer programs, were used to compare the relative effectiveness of dinucleotides containing nitr ogen bases other than nicotinamide or adenine to that of NAD. Effectiv e substrate-competitive inhibition of the pyrophosphatase was observed with purine and pyrimidine nucleoside diphosphates in the low micromo lar concentration range. Although less effective, N-1-alkylnicotinamid e chlorides also inhibited competitively with respect to the substrate , NAD. In addition to being an effective inhibitor of the purified enz yme, adenosine diphosphate also inhibited growth of H. parasuis at a l ow micromolar concentration. This inhibition of growth correlates well with inhibition of the periplasmic pyrophosphatase which is supported by thr fact that adenosine diphosphate does not effectively inhibit g rowth when the pyrophosphatase is by-passed by growth on nicotinamide mononucleotide. These observations are all consistent with the peripla smic nucleotide pyrophosphatase being essential for the growth of the organism on NAD and therefore, a very important enzyme with respect to the pathogenesis of the organism. 3-Aminopyridine mononucleotide, whi ch also inhibited growth of H. parasuis at a low micromolar concentrat ion, did not effectively inhibit the purified pyrophosphatase and a di fferent target enzyme needs to be considered to explain growth inhibit ion by this derivative. (C) 1997 Elsevier Science B.V.