Metabolism of water soluble vitamins : studies on enzymes from mung bean (phasseolus radiatus) seedlings, hydrolyzing coenzyme nucleotides

Abstract

The levels of coenzyme nucleotides can be controlled by the availability of the vitamin in the diet, by environmental conditions such as availability of oxygen or metal ions, by regulation of the enzymes involved in their biosynthesis and degradation, or by the availability of the apoenzymes. Evidence in favour of each of these possibilities has been discussed with special reference to pyridine and flavin coenzyme nucleotides. The evidence supporting the hypothesis that degradative enzymes have a regulatory role in coenzyme nucleotide metabolism is not extensive.

Earlier observations from this laboratory have demonstrated the pathway for the biosynthesis of flavin coenzyme nucleotides and the occurrence of the enzymes hydrolysing FMN at pH 5.0 (219) and FAD at pH 7.4 (104). Hydrolysis of FAD at other pH values and the regulation of these activities by metabolites are reported in this thesis.

A crystalline and electrophoretically homogeneous enzyme hydrolysing FAD, NAD and NADH was obtained from mung bean seedlings. The enzyme was isolated by homogenisation of the seedlings, manganous sulfate treatment, ammonium sulfate fractionation, negative adsorption on alumina C -gel and bentonite, acetone fractionation, adsorption and elution from DEAE-cellulose, and crystallisation with ammonium sulfate.

The enzyme functioned optimally at 49 °C and at pH 9.2. The Km for FAD, NAD and NADH were 2 × 10 M, 5 × 10 M and 1 × 10 M respectively. The enzyme did not hydrolyse NADP , ATP, ADP, AMP, FMN, UTP, UDP, UMP, GTP, GDP, GMP, CTP, CDP, CMP, inorganic pyrophosphate, or glycerophosphate at pH 9.2, 7.4 and 5.0.

The enzyme was inhibited by chelating agents, and the EDTA inhibition could be reversed by Zn² and Co² . The apoenzyme could also be specifically reactivated more efficiently by Zn² than by Co² . The reactivated enzyme was similar to the native enzyme in its pH-temperature optimum, affinity for substrate, and inhibition by AMP and ATP.

Unlike the nucleotide pyrophosphatase functioning at pH 7.4 (104), this enzyme was inactivated by GSH and mercaptoethanol without alteration in the locus of attack.

The enzyme functioned with an initial fast rate followed by a linear, second slower rate. The pH and temperature optima and Km for FAD for both rates were identical. The maximum velocity per minute per mg protein at the faster rate was 4200 units and at the slower rate was 1400 units. The second rate occurred due to non linear inhibition of the enzyme by AMP.

AMP non competitively inhibited the enzyme activity. ADP, ATP, NAD and FMN were without effect. Sodium pyrophosphate inhibited the activity while inorganic orthophosphate had no effect. Tartrate, fluoride and molybdate had no effect.

Aging (dilution) of the enzyme resulted in loss of activity, which could be specifically restored by AMP. Higher concentrations of AMP inhibited the activity. The activating concentrations of AMP increased both Vmax and Km for the aged, diluted enzyme.

Urea and guanidine hydrochloride inactivated the enzyme, and 80% inactivation occurred at 8 M urea or 6 M guanidine hydrochloride. Further increase in concentration did not have an additional effect. Renaturation was achieved either by dilution or by passage through a Sephadex column. The renatured enzyme functioned optimally at pH 9.2 and 49 °C. The enzyme functioned at a linear fast rate, and the second slower rate was abolished. AMP below 8 × 10 M had no effect on the enzyme activity. The Vmax and Km for FAD cleavage for the renatured enzyme were:

Vmax = 2800 units/min/mg protein Km = 4 × 10 M

Zinc induced reactivation had no effect on reversible denaturation induced by urea.

Based on these results, a reactivation sequence was proposed. It was postulated that AMP release was the rate limiting step in the initial as well as the second slow rates. In the AMP activated and the urea renatured enzyme, the rate limiting step was assumed to be the interaction with the substrate. Addition of ATP interfered with AMP release and hence functioned as a linear non competitive inhibitor.

A nucleotide pyrophosphatase functioning at acidic pH was obtained from homogenates of mung bean seedlings by ammonium sulfate fractionation, heat treatment, and negative adsorption on either phosphocellulose or CM cellulose. This enzyme was essentially free of FMN hydrolase and alkaline nucleotide pyrophosphatase activities.

The enzyme functioned optimally at pH 4.0 and at 37-50 °C. The Km and Vmax for FAD were 3 × 10 M and 80 units/min/mg protein, respectively. The initial burst of activity (30 s) was followed by a linear rate up to 7 minutes. The enzyme hydrolysed, at pH 5.0, those nucleotides at 50% the rate of pyrophosphate cleavage.

Pyrophosphate hydrolysis was activated at low concentrations of AMP and inhibited at high concentrations. A similar activation pattern was observed for FAD hydrolysis.

Pyrophosphate and FAD hydrolysis were inherent properties of the same enzyme protein, as indicated by identical temperature and pH optima, sensitivity to heat inactivation, and identical elution patterns on DEAE-cellulose, Sephadex G 100 and Biogel P 200.

The enzyme was not inactivated by EDTA, tartrate, fluoride, pCMB or GSH.

Urea below 4 M concentration had no appreciable effect.

Conclusions Based on the results of these investigations, the following scheme (Fig. 37) of reactions is proposed for the regulation of flavin coenzyme nucleotide levels in plant tissues. Flavokinase is the first enzyme in the biosynthesis of flavin coenzymes from riboflavin. The reaction is essentially irreversible and specific for the substrates riboflavin and ATP. The second enzyme, FAD synthetase, is a reversible reaction generating pyrophosphate and FAD in the forward direction. The enzyme cleaving FAD at alkaline pH, reported in this thesis, does not cleave ATP, ADP, AMP or pyrophosphate, but is inhibited by these metabolites involved in the biosynthetic or degradative sequence. AMP formed during the initial stages of hydrolysis of FAD slows down the hydrolysis. ATP, which is a substrate for both biosynthetic reactions, inhibits in a linear non competitive manner, presumably by inhibiting the rate limiting AMP release. This control would permit biosynthesis to proceed uninterrupted. The nonspecific pyrophosphatase probably has a scavenging function. Even this enzyme is inhibited by AMP, and significantly, AMP inhibits the phosphatase cleaving FMN (95). The work of Tappel and co workers suggests that acid pyrophosphatase is lysosomal in origin (103, 171, 172). Compartmentation of these enzymes provides an additional mechanism of control. These investigations also permit speculation that loss of control of riboflavin biosynthesis in microorganisms excreting flavins may arise due to desensitisation of FAD hydrolysis to AMP inhibition. Because FAD hydrolysis is extremely rapid, the product of flavinogenesis is riboflavin. Further kinetically oriented molecular studies-especially active site labelling and conformational analyses-would clarify the mechanisms of pyrophosphate bond cleavage and the relationship between enzyme conformation and activity.