BIOSYNTHESIS AND METABOLISM OF PYRAZOLE BY CUCUMIS-SATIVUS - ENZYMIC CYCLIZATION AND DEHYDROGENATION OF 1,3-DIAMINOPROPANE

Both 1,3-diaminopropane and pyrazole stimulate synthesis of β-pyrazol-1-ylalanine in seedlings and excised shoots of cucumber. With either plant system, increasing the concentration of pyrazole raised β-pyrazol-1-ylalanine yield to a maximum beyond which increasing inhibition occurred. Similar effects were seen with 1,3-diaminopropane when supplied to excised shoots, but with germinating seedlings, β-pyrazol-1-ylalanine synthesis continued to increase up to the highest concentration of 1,3-diaminopropane presented. An enzymic fraction was obtained which catalysed the formation of pyrazole from 1,3-diaminopropane. The activity was partially purified by fractional precipitation with ammonium sulphate followed by gel-filtration chromatography on Sephadex G-100. A 6.5-fold purification was obtained. The activity was stimulated by FAD but was not affected by NAD+ or NADP+. It was also unaffected by dialysis against buffered EDTA and, by a range of metal ions (except Ca2+ which caused significant inhibition). A sharp optimum was observed at pH 6.2 with little detectable activity above pH 6.5. Gel-filtration chromatography on Sephadex G-100 showed a major and a minor peak of activity; their apparent Mr are 66 000 and 12 600, respectively. Both 1,3-diaminopropane, the precursor of pyrazole, and 2-pyrazoline, a putative intermediate in the biosynthesis of pyrazole from 1,3-diaminopropane, were shown to dilute substantially the radioactivity incorporated from [3,4-14C]pyrazole into β-pyrazol-1-ylalanine. That the isotope dilution effect of 2-pyrazoline is not merely attributable to inhibition of the incorporation of pyrazole into β-pyrazol-1-ylalanine was shown by a parallel experiment in which O-acetyl-[3-14C]serine and unlabelled pyrazole were supplied in the presence and absence of 2-pyrazoline. Studies of the metabolism of [3,4- 14C]pyrazole by cucumber seedlings indicated that in addition to the major products β-pyrazol-1-ylalanine and its γ-glutamyl peptide, four minor metabolites arise. Evidence was obtained that β-pyrazol-1-ylalanine can be further metabolized by Cucumis sativus. © 1990.