PubMed · 11941332
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Abraham M Blechman. 2002. Functional magnetic system.. https://doi.org/10.1067/mod.2002.123337
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Cobalt influences the methanol metabolism of Desulfofundulus kuznetsovii TPOSR, specifically by modulating the activity of one of its alcohol dehydrogenases (ADH), Adh1. However, the effects of cobalt on the broader proteome of strain TPOSR, as well as the utilization of alcohols besides methanol, remain unexplored. Here, proteomic analyses of strain TPOSR grown with and without cobalt on different alcohol substrates show that cobalt starvation impacts multiple cellular processes, including cobalamin biosynthesis, iron-sulphur cluster assembly and, most prominently, energy metabolism as indicated by altered abundances of hydrogenases and NAD(P)-dependent oxidoreductases. Despite the presence of six ADH-encoding genes in the genome, Adh1 is the dominant ADH during growth not only on methanol but also on several primary alcohols and diols (ethanol, 1-propanol, 1,2-propanediol, 1,3-propanediol, butanol, pentanol and heptanol). Enzymatic assays with purified Adh1 confirm activity with these substrates, except 1,3-propanediol, and show no activity toward secondary alcohols (2-propanol and 2-butanol). Comparative proteomics analyses of other sulphate-reducing microorganisms (SRMs), namely Desulfofundulus australicum and Solidesulfovibrio carbinolicus, further indicate that methanol and ethanol oxidation in SRMs is mediated by a single ADH/AOR pair. Together, these findings highlight the central role of cobalt in alcohol metabolism in strain TPOSR and identify conserved ADH/AOR enzymes as promising candidates for biotechnological applications.
The effects of transition metal ions (M2+) such as Mn2+, Co2+, Ni2+, and Cu2+ on the functional and structural stabilities of horseradish peroxidase (HRP) were investigated with respect to reversible chemical denaturation, Michaelis-Menten kinetics, chemical modification and time-dependent catalytic activity. Conformational Gibbs free energy (deltaGo(H2O)) as a structural stability criterion and transition concentrations of metal ions ([M2+] 1/2) were estimated using a two-state chemical denaturation model. Activation and inhibitory concentration ranges for each metal ion were specified by the steady-state enzyme kinetics. Results of a pH-profile method confirmed by chemical modification indicate that a histidine residue interacts in the activation concentration range, whereas carboxylic residues (Asp and Glu) contribute to interaction in the inhibitory concentration range. Incubation of the enzyme with the metal ion at activation concentration leads to long-term functional stability of peroxidase. Thus, such metal ions as potent effectors induced the enhancement of conformational and functional stabilities of horseradish peroxidase.
Isotope effects in the nucleophile and in the leaving group were measured to gain information about the mechanism and transition state of the hydrolysis of methyl p-nitrophenyl phosphate complexed to a dinuclear cobalt complex. The complexed diester undergoes hydrolysis about 1011 times faster than the corresponding uncomplexed diester. The kinetic isotope effects indicate that this rate acceleration is accompanied by a change in mechanism. A large inverse 18O isotope effect in the bridging hydroxide nucleophile (0.937 +/- 0.002) suggests that nucleophilic attack occurs before the rate-determining step. Large isotope effects in the nitrophenyl leaving group (18Olg = 1.029 +/- 0.002, 15N = 1.0026 +/- 0.0002) indicate significant fission of the P-O ester bond in the transition state of the rate-determining step. The data indicate that in contrast to uncomplexed diesters, which undergo hydrolysis by a concerted mechanism, the reaction of the complexed diester likely proceeds via an addition-elimination mechanism. The rate-limiting step is expulsion of the p-nitrophenyl leaving group from the intermediate, which proceeds by a late transition state with extensive bond fission to the leaving group. This represents a substantial change in mechanism from the hydrolysis of uncomplexed aryl phosphate diesters.