Eduardo Cadenas

A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity

Abstract 1. 1. Halobacterium halobium has two chromatographically distinct forms of glutamate dehydrogenase which differ in their thermolability and other properties. One glutamate dehydrogenase utilizes NAD, the other NADP as a coenzyme. 2. 2. The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) was purified 65-fold from crude extracts of H. halobium. 3. 3. The Michaelis constants for 2-oxoglutarate (13.3 mM), ammonium (3.1 mM) and NADPH (0.077 mM) indicate that the enzyme catalyzes in vivo the formation of glutamate from ammonium and 2-oxoglutarate. 4. 4. The amination of 2-oxoglutarate by NADP-specific glutamate dehydrogenase is optimal at the pH value of 8.0–8.5. The optimal NaCl or KCl concentration for the reaction is 1.6 M. 5. 5. None of the several metabolites tested for a possible role in the regulation of glutamate dehydrogenase activity appeared to exert an appreciable influence on the enzyme. 6. 6. NAD- and NADP-dependent glutamate dehydrogenases from H. halobium showed apparent molecular weights of 148,000 and 215,000 respectively.

Purification and some properties of NAD+-dependent glutamate dehydrogenase from Halobacterium halobium

Abstract 1. 1. A NAD + -dependent glutamate dehydrogenase (EC 1.4.1.2.) was purified 126-fold from Halobacterium halobium . 2. 2. Activity and stability of the enzyme were affected by salt concentration. Maximum activity of the NADH-dependent reductive amination of 2-oxoglutarate occurs at 3.2 M NaCl and 0.8 M KCl, and the NAD + -dependent oxidative deamination of l -glutamate occurs at 0.9 M NaCl and 0.4 M KCl. The maximum activity is higher with Na + than with K + in the amination reaction while the reverse is true in the deamination reaction. 3. 3. The apparent K m values of the various substrates and coenzymes under optimal conditions were: 2-oxoglutarate, 20.2 mM; ammonium, 0.45 M; NADH, 0.07 mM; l -glutamate, 4.0 mM; NAD + , 0.30 mM. 4. 4. No effect of ADP or GTP on the enzyme activity was found. The purified enzyme was activated by some l -amino acids.

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

Abstract 1. 1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium. 2. 2. The enzyme has an apparent molecular weight of 24,000. 3. 3. A Km value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions. 4. 4. The enzyme is selectively activated and stabilized by Mn2+. 5. 5. It requires high salt concentrations for stability and maximum activity. 6. 6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

An extreme halophilic enzyme active at low salt in reversed micelles

Possible biotechnological applications of extreme halophilic enzymes are strongly determined by their high salt requirement of around 4 M NaCl. Consequently, the use of these in organic media seemed to be unlikely. However, we have succeeded in dissolving a halophilic enzyme, p-nitrophenylphosphate phosphatase from the archaeon Halobacterium salinarum, in an organic medium by creating a reverse micellar system with very low salt concentration. The enzyme retained its catalytic properties in reversed micelles made with an anionic surfactant (dioctyl sodium sulphosuccinate) or with a cationic surfactant (hexadecyltrimethylammonium bromide) in cyclohexane plus 1-butanol as co-surfactant. The dependence of the rate of hydrolysis of p-nitrophenylphosphate phosphate on the molar water/surfactant ratio (w(0) value) showed a bell-shaped curve for each surfactant system. Kinetic parameters were determined in each system. The enzymatic reaction appeared to follow Michaelis-Menten kinetics with the anionic surfactant only. The kinetic behaviour was determined at different concentrations of Mn(2+) in reversed micelles of dioctyl sodium sulphosuccinate as surfactant.

Alkaline p-nitrophenylphosphate phosphatase activity from halo bacterium halobium. seletive activation by manganese and effect of other divalent cations

1. Alkaline p-nitrophenylphosphate phosphatase (pNPPase) activity of Halobacterium halobium is selectively stabilized and stimulated by Mn2+ ions. 2. Mn2+ binding to native pNPPase is characterized by a dissociation constant of 0.35 mM at pH 8.5, 37 degrees C, with a Hill coefficient of 0.988. 3. Mn2+ behaves as a mixed type nonessential activator, increasing the Vmax value (beta = 6.09, pH 8.5) and decreasing the Km value for pNPP (alpha = 0.56, pH 8.5). The Ki value for inorganic phosphate (a competitive inhibitor) was also decreased in the presence of Mn2+. 4. Activation of native pNPPase by preincubation with Mn2+ is a slow temperature-dependent process, which can be described by an exponential relationship vs time. However, a weak but immediate activation was also detected. 5. Zn2+, Cu2+ and Ni2+ were found to inhibit both native and Mn(2+)-stimulated pNPPase, whereas Co2+ and Cd2+ inhibited the Mn(2+)-stimulated pNPPase but had no effect on the native enzyme form.

Stability of an extreme halophilic alkaline phosphatase from Halobacterium salinarium in non-conventional medium.

Alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium) was solubilised in organic medium using reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant. The stability of alkaline p-nitrophenylphosphate phosphatase in this system was studied at different conditions, w(0) ([H(2)O]/[surfactant]), salt concentration, with and without Mn(+2). At all the conditions assayed, alkaline p-nitrophenylphosphate phosphatase was more stable in reversed micelles than in bulk aqueous solution (at 25 degrees C). The stabilisation effect of the reversed micelles was dramatic when the enzyme was dialysed against Mn(+2)-free buffer since the enzyme lost all the activity within 90 min in aqueous medium, but it retained approximately 72% of the initial enzymatic activity for 90 min in reversed micelles.

ANALYSIS OF THE KINETIC MECHANISM OF HALOPHILIC NADP-DEPENDENT GLUTAMATE DEHYDROGENASE

The amination of 2-oxoglutarate catalyzed by NADP-specific glutamate dehydrogenase (EC 1.4.1.4, L-glutamate:NADP+ oxidoreductase (deaminating)) from Halobacterium halobium has been analyzed by initial rate, graphical analysis, and product and competitive inhibition studies. Initial rate and graphical analysis reveal that a B term (representing 2-oxoglutarate) is not statistically necessary for an initial rate equation. However, the absence of a B term does not distinguish between ordered and random binding of NADPH and ammonia. The patterns of product inhibition by NADP+ and L-glutamate, and competitive inhibition by hydroxylamine and succinate permit deduction of the kinetic mechanism as ordered, with NADPH, 2-oxoglutarate and ammonia added in that order, and L-glutamate release preceding NADP+ release.

Kinetic Studies of an Extremely Halophilic Enzyme Entrapped in Reversed Micelles

Alkaline p-nitrophenylphosphate phosphatase (pNPPase) from the halophilic archaeobacterium Halobacterium salinarum (earlier halobium) was solubilised in reversed micelles of hexadecyltrimethylammoniumbromide (CTAB) in cyclohexane, with 1-butanol as co-surfactant. The hydrolysis of p-nitrophenylphosphate (pNPP) appeared to follow Michaelis-Menten kinetics. Km and Vmax depended on the method of reaction initiation. The kinetic parameters of halophilic pNPPase in CTAB reversed micelles with high salt concentration (0.85 M NaCl) were determined. pNPPase showed the same dependency on the buffer ionic strength in reversed micelles as in aqueous macrosolution. The dependence of the maximum reaction rate (Vmax) on the molar water/surfactant ratio (ω0 value) showed a bell-shaped curve for NaCl and KCl, with a maximum reaction rate being found at ω0 = 10.27. The pH value for the maximum reaction rate was 9.0. The optimum temperature for enzyme activity was around 45°C.

Kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase

The kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase (EC 1.4.1.3) has been investigated at pH 9.0, 3 M NaCl and 40 degrees C in both directions, by initial rate and inhibition studies. The results of the initial rate studies indicate that the mechanism is sequential with respect to substrate addition. The inhibition patterns obtained with halophilic NAD+-glutamate dehydrogenase are not consistent with a simple ordered mechanism without modification. They can, however, be reconciled with this type of mechanism by postulating an appropriate abortive complex.

The dependence of a halophilic malate dehydrogenase on ωo and surfactant concentration in reverse micelles

Abstract The halophilic malate dehydrogenase (hMDH) activity of Halobacterium salinarum was studied as a function of micelle size (ωo), in cethyltrimethylammonium bromide (CTAB)/cyclohexane reverse micelles, with 1-butanol as cosurfactant. The velocity dependence of the ωo profile depends on the buffer used, the surfactant concentration, and the salt concentration. In phosphate buffer, the activity increases with increasing water content, while in Tris/HCl buffer a bell-shaped profile is generally observed. Despite a slight change in the ωo-activity profile, the enzymatic activity was higher at low salt concentration even when we employed a different buffer. The ωo value for the maximal activity (optimum ωo) varies directly with the enzyme concentration. The hMDH activity in reverse micelles depends on the surfactant concentration and the dependence of the activity of this enzyme on the surfactant concentration, at constant ωo, is different for each ωo value.