Search

Human myristoyl-CoA:protein N-myristoyltransferase (hNmt) catalyzes the transfer of myristate from CoA to the amino-terminal Gly residue of a number of cellular proteins involved in signal transduction pathways, to structural and nonstructural proteins encoded by retroviruses, hepadnaviruses, picornaviruses, and reoviruses, as well as to several transforming tyrosine kinases. hNmt has been purified 230-fold from an erythroleukemia cell line. The monomeric enzyme has no associated methionyl aminopeptidase activity. To determine the enzyme's kinetic mechanism, we examined the effect of covariation of subsaturating concentrations of myristoyl-CoA and peptide substrate on initial velocity. Double-reciprocal plots excluded a double displacement (ping-pong) mechanism. Product inhibition studies indicated that CoA was a noncompetitive inhibitor against myristoyl-CoA and a mixed-type inhibitor against peptide substrates. Together these results are consistent with a sequential ordered mechanism where, in a typical catalytic cycle, myristoyl-CoA binds to apoenzyme before peptide followed by release of the CoA and then myristoylpeptide products. This kinetic mechanism is identical to that described for Saccharomyces cerevisiae N-myristoyl-transferase (Nmt1p) and emphasizes the impact that regulation of myristoyl-CoA pool size and accessibility may have in modulating protein N-myristoylation in these two species. Comparative studies of the peptide substrate specificities of hNmt and Nmt1p using a panel of 12 octapeptides revealed distinct differences in their tolerance for amino acid substitutions at positions 3, 4, 7, and 8 of parental peptides derived from the amino-terminal sequences of known N-myristoyl-proteins. This finding contrasts with our recent observation that the acyl-CoA substrate specificities of hNmt and Nmt1p are highly conserved and suggests that these differences in peptide recognition provide an opportunity to develop species-specific enzyme inhibitors.

An analysis of the effect of DNA supercoiling on transcription of the RNA-I promoter of ColE1 has been undertaken using the abortive initiation assay developed by McClure (McClure, W. R., Cech, C. L., and Johnston, D. E. (1978) J. Biol. Chem. 253, 8941-8948). A tau plot was constructed, enabling the equilibrium constant for closed complex formation (KI) and the isomerization rate constant (k2) to be determined. The effect of supercoiling on RNA-I is an apparent 100-fold increase in the isomerization rate, k2, with little or no effect on KI. This is the first reported abortive initiation analysis of RNA-I transcription which serves in the regulation of plasmid replication.

The pH-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been more extensively characterized. The native, dimeric form of the enzyme (Mr = 70,000) which exists at pH 7.5 has previously been shown to dissociate into its constituent subunits (Mr = 35,000) at pH 5.0 (Bleile, D. M., Schulz, R. A., Gregory, E. M., and Harrison, J. H. (1977) J. Biol. Chem. 252, 755-758). The dissociation is accompanied by a concomitant decrease in enzymatic specific activity and an increase in intrinsic protein fluorescence. By using the characteristics of specific activity and intrinsic protein fluorescence as probes of dimerization, the kinetics of subunit reassociation was investigated. In order to facilitate reassociation, a pH jump method was utilized in which enzyme at pH 5.0 was diluted into a large excess of pH 7.5 buffer. The regain of enzymatic specific activity and the decrease in protein fluorescence were observed to follow first order kinetics. The rate constant in both cases was dependent upon the protein concentration, and in all cases, full recovery of either enzymatic activity or native protein fluorescence was obtained. The Arrhenius activation energy for the reassociation of the subunits was found to be approximately 20 kcal/mol, an observation which is consistent with a refolding process whose rate-limiting step may be the cis/trans-isomerization about one or more proline imino bonds. A model for subunit reassociation which is consistent with the kinetic data is proposed.

An analysis of the effect of DNA supercoiling on transcription of the RNA-I promoter of ColE1 has been undertaken using the abortive initiation assay developed by McClure (McClure, W. R., Cech, C. L., and Johnston, D. E. (1978) J. Biol. Chem. 253, 8941-8948). A tau plot was constructed, enabling the equilibrium constant for closed complex formation (KI) and the isomerization rate constant (k2) to be determined. The effect of supercoiling on RNA-I is an apparent 100-fold increase in the isomerization rate, k2, with little or no effect on KI. This is the first reported abortive initiation analysis of RNA-I transcription which serves in the regulation of plasmid replication.

Porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been immobilized by covalent attachment to CNBr-activated Sepharose 4B-Cl gel. The gel was activated with low levels of CNBr to produce a low density of linkage sites and, hence, to facilitate linkage of the enzyme through a single subunit. Matrix-bound mitochondrial malate dehydrogenase was found to possess 50-65% of the native mitochondrial malate dehydrogenase specific activity when assayed in the NAD+ leads to NADH direction but only 5-15% of the native enzyme specific activity when assayed in the NADH leads to NAD+ direction. MB-dimeric mitochondrial malate dehydrogenase was dissociated to MB-monomer by exposure to pH 5.0 buffer. The MB-monomer was found to be catalytically active, possessing only a slightly decreased specific activity when compared to MB-dimer. The reconstitution of Mb-monomer to MB-dimer was accomplished by adding dissociated mitochondrial malate dehydrogenase, which exists at pH 5.0, to MB-monomer and adjusting to pH 7.5. The kinetic parameters, pH activity profile, and stability toward heat denaturation for MB-mitochondrial malate dehydrogenase (monomer and dimer) were determined and compared to native mitochondrial malate dehydrogenase. MB-mitochondrial malate dehydrogenase exhibited enhanced stability and similar pH activity profiles when compared to native mitochondrial malate dehydrogenase. Immobilization of mitochondrial malate dehydrogenase altered the enzyme's kinetic parameters in such a manner as to increase the values of Km for the substrates and decrease the values of Vmax.

The specific chemical modification by N-ethylmaleimide of a cysteine residue at pH 5.0 in porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been shown to result in an enzymatically inactive, monomeric product, which does not reassociate at pH 7.5 to yield the native dimer. In this report, an investigation of proton release and uptake upon NADH binding to the native enzyme and to the N-ethylmaleimide-modified enzyme has implicated the above cysteine residue as being directly linked to the pH-dependent subunit dissociation of mitochondrial malate dehydrogenase. The results are consistent with the view that the modified cysteine residue is not located at the subunit interaction site, although it is probably near this site. A recent study from this laboratory has demonstrated that the monomeric enzyme obtained at pH 5.0 exists in a conformation which is enzymatically inactive and which has an enhanced intrinsic protein fluorescence. Interpretation of protein fluorescence data has suggested that the N-ethylmaleimide modification results in inactivation of the enzyme by preventing the pH-induced conformational change to the active dimer. However, NADH is able to induce reassociation of the N-ethylmaleimide-modified enzyme at pH 7.5 but not at pH 5.0. This reassociation at pH 7.5 is accompanied by a significant regain of enzymatic activity, indicating that NADH binding is able to partially overcome the negative effect of the cysteine modification on the pH-dependent subunit reassociation of mitochondrial malate dehydrogenase.

The pH-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been more extensively characterized. The native, dimeric form of the enzyme (Mr = 70,000) which exists at pH 7.5 has previously been shown to dissociate into its constituent subunits (Mr = 35,000) at pH 5.0 (Bleile, D. M., Schulz, R. A., Gregory, E. M., and Harrison, J. H. (1977) J. Biol. Chem. 252, 755-758). The dissociation is accompanied by a concomitant decrease in enzymatic specific activity and an increase in intrinsic protein fluorescence. By using the characteristics of specific activity and intrinsic protein fluorescence as probes of dimerization, the kinetics of subunit reassociation was investigated. In order to facilitate reassociation, a pH jump method was utilized in which enzyme at pH 5.0 was diluted into a large excess of pH 7.5 buffer. The regain of enzymatic specific activity and the decrease in protein fluorescence were observed to follow first order kinetics. The rate constant in both cases was dependent upon the protein concentration, and in all cases, full recovery of either enzymatic activity or native protein fluorescence was obtained. The Arrhenius activation energy for the reassociation of the subunits was found to be approximately 20 kcal/mol, an observation which is consistent with a refolding process whose rate-limiting step may be the cis/trans-isomerization about one or more proline imino bonds. A model for subunit reassociation which is consistent with the kinetic data is proposed.

Addition of trans-(Ph3P)2 Ir(CO)Cl to trans-CF3(CN)CC(CF3)CN affords (Ia)(Ph3P)2Ir(CO)Cl[CF3(CN)C2(CF3)CN] where the phosphine ligands and CF3 groups have respectively a relative cis- and trans-configuration. trans-(Ph2PMe)2Ir(CO)Cl reacts with the same olefin to form and isostructural complex (II). Stilbene(bistriphenylphosphine)platinum adds to trans-CF3(CN)CC(CF3)CN to form a vinyl complex (III)(Ph3P)2PtCN-[C(CF3)tC(CN)CF3]. The kinetics of the reaction with trans-(Ph3P)2Ir(CO)Cl to form (Ia) have been studied in different solvents and activation parameters obtained. Reaction of cis-/trans-CF3(CN)CC(CF3)CN with trans-(Ph3P)2Ir(CO)Cl gives only (Ia), and this is interpreted in terms of an ionic-intermediate. The formation of (Ph3P)2Pt[CF(Cl)C(F)Cl](IVa)(trans) and (Ph3P)2Pt[CF(Cl)C(Cl)F](IVb)(cis), which differ only in the stereochemistry of the olefin, in the ratio of trans : cis= 2·3 : 1 from, stilbenebis(triphenylphosphine)platinum and 1,2-dichloro-1,2-difluoroethylene (cis : trans= 1 : 1), is also interpreted in terms of a similar ionic-intermediate.