Porphyrin biosynthesis in Rhodopseudomonas palustris—IX. PBG-deaminase. Kinetic studies

Abstract 1. 1. PBG-Deaminase obtained from Rp. palustris exhibited classical Michaelis-Menten kinetics in the absence or presence of different ions. 2. 2. Detailed kinetic studies were carried out in the presence of ammonium, phosphate and magnesium ions. 3. 3. It has been found that the different effects observed are dependent on both the substrate and the ion concentration.

PBG-Deaminase from Rhodopseudomonas palustris, the most common non sulfur bacteria, has also been purified by Kotler et al. (1986). The present work describes kinetic studies conducted on PBG-Deaminase in the absence and presence of different ions.

MATERIALS AND METHODS
Porphobilinogen was biosynthetically obtained (Sancovich et al., 1970) and estimated as described by Moore and Labbe (1964). The standard incubation system contained the enzyme preparation (50 pl) together with 0.05 M sodium phosphate buffer (pH 7.6-7.8) and PBG (at the concentrations indicated) with or without the addition of other reagents, in a final volume of 1.5 ml. Incubations were carried out aerobically in the dark with mechanical shaking at 37°C for 30 min.
Blanks were always run with PBG and without enzyme.
After incubation, TCA was added to precipitate the protein (final concentration 5% w/v), the mixture was then exposed to white light for 20 min to oxidize porphyrinogens, the protein precipitate filtered off and total porphyrins and remaining PBG estimated in the resulting solution (Rimington, 1960;Moore and Labbe, 1964).
In all experiments reaction velocity was measured on the basis of Uroporphyrinogen formation and substrate consumption during 30 rain or 1 hr.
The purification procedure for PBG-Deaminase, as well as all other methods and materials not specified here, were those described by Kotler et al. (1986).

RESULTS
Saturation curves (Fig. 1) Plots of velocity measured as nmol of uroporphyrinogen formed against PBG concentration and their reciprocals followed classical Michaelis-Menten kinetics.
When velocity was measured in terms of PBG consumed, the saturation curve was linear and the double reciprocal plots also showed a normal kinetic pattern.
Michaelis constants and maximum velocities were calculated from Lineweaver-Burk plots and the number of probable substrate binding sites (n) were determined by the empirical Hill equation (Fig. l,  inset).
In Euglena graeilis Williams et al. (1981) demonstrated that PBG-deaminase combined four molecules of substrate to produce one molecule of uroporphydnogen I with the elimination of four molecules of ammonia, this stoicheiometry was observed at different pH values (range 5.5-8.5).
In our laboratory, working with PBG-ase from soya-bean callus (Llambias and Batlle, 1971), Euglena gracilis (Rossetti, 1978) and Rp. palustris (Juknat, 1983) we have shown that uroporphyrinogen formation based on substrate consumption was extremely low, indicating a great deviation from stoicheiometric values. When PBG-deaminase from Rp. palustris was studied, an excess of PBG consumption M. L. KOTLER et al.  of 5 times at low substrate concentration (8-26/~ M) was found, increasing this value up to 50 times at 350#M PBG (Table 1). Battersby et al. (1983a) reported an inhibitory effect of hydroxymethylbilane on PBG-Deaminase activity from E. gracilis, a fact that could be an explanation for these findings.

Effect of magnesium ions
The the affinity for PBG. At the same time there was a decrease in Vm~ tending to 2 nmol/hr. Inhibition of PBG-deaminase activity by high substrate concentration at 50 and 100 mM Mg 2+ was also found. It has also been observed that at 25, 50 and 100/~M PBG, low con~ntration of Mg 2+ activated the enzyme (Fig. 3). Increasing Mg 2+ concentrations however produced a great inhibition, reaching 100% for 100mM Mg 2+ and 500/~M PBG.
Mg 2+ ions had no effect on PBG consumption.

Effect of phosphate ions
Activity plots of PBG-deaminase vs PBG concentration at different PO4 ~-concentrations, showed an hyperbolic pattern. Reciprocal plots (Fig. 4) and Eadie curves were also linear. From the reciprocal plots, it can be seen that at 1 and 10 mM, phosphate behaved as an incompetitive inhibitor.
When velocity of PBG-deaminase against PO4 3concentrations was plotted, typical inhibition curves were obtained (Fig. 5). The experiments were performed as described in the text. ~?.alculated on the basis of the PBG uptake. bR: nmol theoretic porphyrins/nmol porphyrins really formed It is interesting to note that if PBG consumption was taken as a measure of enzyme activity, velocity plots of PBG-deaminase against PBG concentration showed normal kinetic pattern at all PO 3-concentrations studied (Fig. 6). It was also found that in this case PO 3-acted as a non-competitive inhibitor.

Effect of ammonium ions
Direct and reciprocal plots of reaction velocity against PBG concentration, both in the absence and the presence of NH~ ions, showed classical Michaelis-Menten kinetics (Fig. 7). Eadie plots were also linear.
Ammonium ion is a non competitive inhibitor of porphyrin synthesis, resulting in lower values of Km and Vm,x. These results are in agreement with Sancovich et al. (1969) and Llambias and Batlle (1971b).
It was found that ammonium at concentrations up to 100mM inhibited 57% urogen I formation at 310#M PBG (Fig. 8).
However, it is interesting to add that NH~ at concentrations that inhibited porphyrin biosynthesis, had no effect upon the rate of PBG consumption.

DISCUSSION AND CONCLUSIONS
The kinetic experiments showed in this report demonstrated that PBG-deaminase from Rp. palustris had a Michaelis kinetic behavior (Fig. 1).
The action of Mg 2+ on PBG-deaminase activity showed that this metal had two different effects, acting as activator or as a non-competitive inhibitor at low and high concentrations respectively. The existence and extent of these effects were found to be dependent on PBG concentration (Fig. 3).
It must also be noted here that Mg 2+ ions did not change normal kinetic pattern.
Studying the effect of Mg :+ on PBG-ase activity of the same source it was observed that 50 mM Mg ~+ also produced activation when 24#M PBG was  employed as substrate concentration . The activating effect of certain concentrations of Mg 2+ could be explained as if they were producing some association-dissociation phenomena, which gives the enzyme a definitive structural arrangement necessary for maximal activity (Batlle and Rossetti, 1977). We proposed that magnesium could act by binding at different sites. At low concentration of Mg 2+, this ion will act producing the optimum structural arrangement, as already suggested. By increasing its concentration, Mg 2+ could inhibit blocking the tetrapyrrole liberation on the PBG-deaminase.
Studies carried out on PBG-deaminase in the presence of phosphate ions, showed that PO]-was acting as an inhibitor of velocity in terms of porphyrin synthesis and PBG consumption, without changes in the kinetic pattern.
It was suggested (Batlle and Rossetti, 1977;Pollack and Russell, 1978) that certain groups (e-amines, imidazoles or guanidines) would be involved in the binding of PBG to PBG-deaminase. It is possible that phosphate could act partially or totally neutralyzing these recognition sites. So, complexs like E-PBG-I and E-I between the enzyme (E), the substrate (PBG) and the inhibitor (I) could be formed. The noncompetitive type of inhibition found in these experiments, could be explained by the formation of this kind of complex.
Taking into account these hypothesis, once produced these complexs, the polypyrrol chain could not reach the length of a tetrapyrrol. So, the PBG consumption diminished, without being reversed by increasing substrate concentration. As a consequence, the synthesis of porphyrins will also be inhibited.
Increasing PO4 ~ concentration, a change in the type of inhibition for porphyrin synthesis can be observed. This effect may be due to the existence of an interaction phenomena between PO~-and porphyrins, which could modify negatively the rate of porphyrin liberation.
Results obtained studying the kinetics of PBGdeaminase in the presence and the absence of ammonium ions, showed classical Michaelis-Menten behaviour and n was near 1. We also found that PBG consumption was not affected, while porphyrin synthesis was inhibited by ammonium ions. Taking into account these results, we suggested that ammonium has no binding site on the PBG-deaminase but produces a decrease in the amount of available tetrapyrroles. Basic derivates could be formed by direct reaction of NH2-with di and tripyrrylmethanes (Davies and Neuberger, 1973). So, polypyrrolic intermediates will be liberated and the tetrapyrrylmethane (TPM) concentration on the PBG-Deaminase will be in turn reduced.