Indoeacetaldoxime hydro-lyase of gibberella fujikoroi

Abstract

In the following pages, the work described in the preceding chapters has been summarized.

The conversion of IAOX to IAN was shown to be an enzymatic process.

Culture conditions for the growth of Gibberella fujikuroi were established. It was found that growing G. fujikuroi at lower temperatures (19–23?°C) elicited maximum IAOX hydro?lyase activity. At higher temperatures (25–30?°C), G. fujikuroi lost its capacity to exhibit IAOX hydro?lyase activity upon continuous sub?culturing at these temperatures.

Assay methods for the estimation of IAOX and IAN have been described.

An extraction procedure for IAOX hydro?lyase from G. fujikuroi has been described. Removal of a precipitate sedimenting upon high?speed centrifugation (12,000?×?g) from the crude extract was found to be necessary for complete recovery of enzyme activity.

IAOX hydro?lyase was purified ~25?fold with ~20% recovery by ammonium sulfate fractionation and column chromatography on DEAE?cellulose. The enzyme was fairly stable and could be stored frozen for 2–10?weeks without much loss of activity.

The general properties of the enzyme were studied. The pH optimum for the enzyme at 30?°C is 7.0, and the enzyme is maximally stable between pH 6.0 and 7.0. The reaction obeyed Michaelis–Menten kinetics at lower substrate concentrations, and the Km for the enzyme with IAOX as substrate was calculated to be about 1.7?×?10???M.

The optimum temperature for the IAOX hydro?lyase reaction was found to be 30–40?°C, and the temperature coefficient (Q10) between 10 and 30?°C was ~2.0. The activation energy was calculated to be ~15,970?cal?mol?¹.

The reaction has been shown to be a dehydration type, and stoichiometric amounts of IAN were formed from IAOX.

The enzyme appears to be highly specific for IAOX, since no other oxime tested was attacked by IAOX hydro?lyase.

However, several aldoximes having structures similar to IAOX inhibited the IAOX hydro?lyase reaction. Inhibition by PAOX (possessing a similar two?carbon side chain as in IAOX) was found to be competitive in nature. The Ki for PAOX was 2.2?×?10?³?M, indicating a very high affinity of PAOX for the enzyme.

The enzyme was inhibited by heavy metal ions such as Hg²?, Cu²?, etc., indicating sulfhydryl involvement in the enzyme. This was also indicated by the inhibitory effects of sulfhydryl reagents such as p?chloromercuribenzoate (PCMB/PMB), iodoacetamide, phenyl isocyanate, and arsenite on IAOX hydro?lyase activity.

Both Fe²? and Fe³? had a stimulatory effect on IAOX hydro?lyase activity. However, activation by Fe³? was not found with DEAE?cellulose–purified enzyme.

Many sulfhydryl compounds such as BAL (dimercaprol), ??mercaptoethanol, cysteine, etc., except glutathione, were powerful inhibitors of IAOX hydro?lyase activity.

The unique nature of the enzyme was also apparent when it was observed that both ascorbic acid and dehydroascorbic acid (DHA) stimulated the enzyme activity. Activation by DHA was apparently very large, but even under anaerobic conditions, ascorbic acid enhanced the activity of the enzyme.

Variably reduced pteroylglutamic acids affected the enzyme reaction differently, whereas folic acid had no effect. THFA (tetrahydrofolic acid) highly activated the enzyme, and DHFA (dihydrofolic acid) inhibited it.

Several reducing agents such as KCN and dithionite inhibited the enzyme. The inhibition by KCN was fully reversed by PLP (pyridoxal?5??phosphate; PALP), whereas the inhibition by dithionite was reversed by DHA. KCN at lower concentrations enhanced IAOX hydro?lyase activity, probably by removing heavy metal ions from the active site of the enzyme.

Of the several pyridoxine derivatives, only PLP (PALP) activated the enzyme. Sodium borohydride reduction did not abolish the activity, indicating that if PLP was bound by a Schiff base (azomethine) linkage, the reduction did not render the enzyme inactive.

A sulfhydryl group requirement was further confirmed by: (a) Inhibition by PCMB (PHMB) which, after removal on Sephadex G?25 gel filtration, was reversed by glutathione; and (b) Protection of IAOX hydro?lyase activity from NEM (N?ethylmaleimide) inhibition by PAOX, a competitive inhibitor.

Involvement of an “oxidized” function in the activity of IAOX hydro?lyase was indicated by inhibition by several reducing agents. This was further confirmed by the facts that inhibition by BAL was fully reversed after removal of excess BAL by gel filtration and incubation with DHA; inhibition was also partially reversed by air?oxidation and by PLP.

The activation by PLP of IAOX hydro?lyase activity appeared to be due to an activation of the catalytic process per se.

The enzyme was inhibited by both catalase and peroxidase. Catalase inhibition was not reversed by cyanide, whereas peroxidase inhibition was reversed by cyanide. Both inhibitions were partially reversed by ascorbic acid or dehydroascorbic acid. Catalase inhibition was also reversed by Fe²?.

Based on the above findings and by analogy with organic reactions, probable mechanisms for the dehydration of IAOX by IAOX hydro?lyase have been considered.