Mutation of H63 and its catalytic affect on the methionine aminopeptidase from Escherichia coli.

In order to gain insight into the mechanistic role of a flexible exterior loop near the active site, made up of Y62, H63, G64, and Y65, that has been proposed to play an important role in substrate binding and recognition in the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H63A enzyme was prepared. Mutation of H63 to alanine does not affect the ability of the enzyme to bind divalent metal ions. The specific activity of H63A EcMetAP-I was determined using four different substrates of varying lengths, namely, l-Met-p-NA, MAS, MGMM and MSSHRWDW. For the smallest/shortest substrate (l-Met-p-NA) the specific activity decreased nearly seven fold but as the peptide length increased, the specific activity also increased and became comparable to WT EcMetAP-I. This decrease in specific activity is primarily due to a decrease in the observed k(cat) values, which decreases nearly sixty-fold for l-Met-p-NA while only a four-fold decrease is observed for the tri- and tetra-peptide substrates. Interestingly, no change in k(cat) was observed when the octa-peptide MSSHRWDW was used as a substrate. These data suggest that H63 affects the hydrolysis of small peptide substrates whereas large peptides can overcome the observed loss in binding energy, as predicted from K(m) values, by additional hydrophilic and hydrophobic interactions.

A deeply divergent phosphoglucomutase (PGM) of Giardia lamblia has both PGM and phosphomannomutase activities.

Giardia lamblia, which is an important parasitic cause of diarrhea, uses activated forms of glucose to make glycogen and activated forms of mannose to make glycophosphosphoinositol anchors. A necessary step for glucose activation is isomerization of glucose-6-phosphate to glucose-1-phosphate by a phosphoglucomutase (PGM). Similarly, a phosphomannomutase (PMM) converts mannose-6-phosphate to mannose-1-phosphate. While whole genome sequences of Giardia predict two PGM candidates, no PMM candidate is present. The hypothesis tested here is that at least one of the two Giardia PGM candidates has both PGM and PMM activity, as has been described for bacterial PGM orthologs. Nondenaturing gels showed that Giardia has two proteins with PGM activity, one of which also has PMM activity. Phylogenetic analyses showed that one of the two Giardia PGM candidates (Gl-PGM1) shares recent common ancestry with other eukaryotic PGMs, while the other Giardia PGM candidate (Gl-PGM2) is deeply divergent. Both Gl-PGM1 and Gl-PGM2 rescue a Saccharomyces cerevisiae pgm1Delta/pgm2Delta double deletion strain, while only Gl-PGM2 rescues a temperature-sensitive PMM mutant of S. cerevisiae (sec53-ts). Recombinant Gl-PGM1 has PGM activity only, whereas Gl-PGM2 has both PGM and PMM activities. We conclude that Gl-PGM1 behaves as a conventional eukaryotic PGM, while Gl-PGM2 is a novel eukaryotic PGM that also has PMM activity.

Oxidative disassembly of the [2Fe-2S] cluster of human Grx2 and redox regulation in the mitochondria.

Mitochondrial Grx2 is a new member of the thioredoxin superfamily that has been found to bind a [2Fe-2S] cluster in a novel coordination motif at the interface of a homodimer, where cluster binding occurs via a catalytic cysteine residue and a molecule of GSH (per monomer). The (Grx2)(2)-[2Fe-2S] dimer is thought to undergo cluster destruction and monomerization in a redox-induced pathway of activation. In this report, we make use of protein film voltammetry (PFV) as a method to probe the stability of the Grx2-[2Fe-2S] cluster, using oxidative poises of varying potential and duration to probe the thermodynamic and kinetic stability of the cluster's electrochemical response. We find that the cluster signal is stable at positive potentials up to 0.5 V but that cluster destruction occurs readily when oxidative pulses in excess of this value are applied.

Analyzing the binding of Co(II)-specific inhibitors to the methionyl aminopeptidases from Escherichia coli and Pyrococcus furiosus.

Methionine aminopeptidases (MetAPs) represent a unique class of protease that is capable of the hydrolytic removal of an N-terminal methionine residue from nascent polypeptide chains. MetAPs are physiologically important enzymes; hence, there is considerable interest in developing inhibitors that can be used as antiangiogenic and antimicrobial agents. A detailed kinetic and spectroscopic study has been performed to probe the binding of a triazole-based inhibitor and a bestatin-based inhibitor to both Mn(II)- and Co(II)-loaded type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs. Both inhibitors were found to be moderate competitive inhibitors. The triazole-type inhibitor was found to interact with both active-site metal ions, while the bestatin-type inhibitor was capable of switching its mode of binding depending on the metal in the active site and the type of MetAP enzyme.

Kinetic and spectroscopic analysis of the catalytic role of H79 in the methionine aminopeptidase from Escherichia coli.

To gain insight into the role of the strictly conserved histidine residue, H79, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli ( EcMetAP-I), the H79A mutated enzyme was prepared. Co(II)-loaded H79A exhibits an overall >7000-fold decrease in specific activity. The almost complete loss of activity is primarily due to a >6000-fold decrease in k cat. Interestingly, the K m value obtained for Co(II)-loaded H79A was approximately half the value observed for wild-type (WT) EcMetAP-I. Consequently, k cat/ K m values decreased only 3000-fold. On the other hand, the observed specific activity of Mn(II)-loaded H79A EcMetAP-I decreased by approximately 2.6-fold while k cat decreased by approximately 3.5-fold. The observed K m value for Mn(II)-loaded H79A EcMetAP-I was approximately 1.4-fold larger than that observed for WT EcMetAP-I, resulting in a k cat/ K m value that is lower by approximately 3.4-fold. Metal binding, UV-vis, and EPR data indicate that the active site is unperturbed by mutation of H79, as suggested by X-ray crystallographic data. Kinetic isotope data indicate that H79 does not transfer a proton to the newly forming amine since a single proton is transferred in the transition state for both the WT and H79A EcMetAP-I enzymes. Therefore, H79 functions to position the substrate by hydrogen bonding to either the amine group of the peptide linkage or a backbone carbonyl group. Together, these data provide new insight into the catalytic mechanism of EcMetAP-I.

Analyzing the catalytic role of Asp97 in the methionine aminopeptidase from Escherichia coli.

An active site aspartate residue, Asp97, in the methionine aminopeptidase (MetAPs) from Escherichia coli (EcMetAP-I) was mutated to alanine, glutamate, and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal-binding site in EcMetAP-I. These mutant EcMetAP-I enzymes have been kinetically and spectroscopically characterized. Inductively coupled plasma-atomic emission spectroscopy analysis revealed that 1.0 +/- 0.1 equivalents of cobalt were associated with each of the Asp97-mutated EcMetAP-Is. The effect on activity after altering Asp97 to alanine, glutamate or asparagine is, in general, due to a approximately 9000-fold decrease in k(ca) towards Met-Gly-Met-Met as compared to the wild-type enzyme. The Co(II) d-d spectra for wild-type, D97E and D97A EcMetAP-I exhibited very little difference in form, in each case, between the monocobalt(II) and dicobalt(II) EcMetAP-I, and only a doubling of intensity was observed upon addition of a second Co(II) ion. In contrast, the electronic absorption spectra of [Co_(D97N EcMetAP-I)] and [CoCo(D97N EcMetAP-I)] were distinct, as were the EPR spectra. On the basis of the observed molar absorptivities, the Co(II) ions binding to the D97E, D97A and D97N EcMetAP-I active sites are pentacoordinate. Combination of these data suggests that mutating the only nonbridging ligand in the second divalent metal-binding site in MetAPs to an alanine, which effectively removes the ability of the enzyme to form a dinuclear site, provides a MetAP enzyme that retains catalytic activity, albeit at extremely low levels. Although mononuclear MetAPs are active, the physiologically relevant form of the enzyme is probably dinuclear, given that the majority of the data reported to date are consistent with weak cooperative binding.

Unraveling the catalytic mechanism of nitrile hydratases.

To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k(cat) and K(m) for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK(E)(S1) and pK(E)(S2) values of 5.9 +/- 0.1 and 9.2 +/- 0.1 (k(cat)' = 130 +/- 1 s(-1)), respectively, and pK(E)(1) and pK(E)(2) values of 5.8 +/- 0.1 and 9.1 +/- 0.1 (k(cat)'/K(m)' = (6.5 +/- 0.1) x 10(3) s(-1) mm(-1)), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 degrees C, an Arrhenius plot was constructed by plotting ln(k(cat)) versus 1/T, providing E(a) = 23.0 +/- 1.2 kJ/mol. The thermal stability of PtNHase also allowed DeltaH(0) ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK(E)(S1) and pK(E)(S2) values. Based on DeltaH(0)(ion) data, pK(E)(S1) is assigned to betaTyr(68), whereas pK(E)(S2) is assigned to betaArg(52), betaArg(157), or alphaSer(112) (NHases are alpha(2)beta(2)-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.

A new colorimetric assay for methionyl aminopeptidases: examination of the binding of a new class of pseudopeptide analog inhibitors.

A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline (L-Met-p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV-Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli (EcMetAP-I) and the type II MetAP from Pyrococcus furiosus (PfMetAP-II). Using L-Met-p-NA, the kinetic constants k(cat) and K(m) were determined for EcMetAP-I and PfMetAP-II and were compared with those obtained with a "standard" high-performance liquid chromatography (HPLC) discontinuous assay. The assay has also been used to determine the temperature dependence of the kinetic constant k(cat) for PfMetAP-II as well as to screen two novel pseudopeptide inhibitors of MetAPs. The results demonstrate that L-Met-p-NA provides a fast, convenient, and effective substrate for both type I and type II MetAPs and that this substrate can be used to quickly screen inhibitors of MetAPs.

The proteomics of N-terminal methionine cleavage.

Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision. According to the generally accepted cleavage rules for MAP, this enzyme cleaves all proteins with small side chains on the residue in the second position (P1'), but many exceptions are known. The substrate specificity of Escherichia coli MAP1 was studied in vitro with a large (>120) coherent array of peptides mimicking the natural substrates and kinetically analyzed in detail. Peptides with Val or Thr at P1' were much less efficiently cleaved than those with Ala, Cys, Gly, Pro, or Ser in this position. Certain residues at P2', P3', and P4' strongly slowed the reaction, and some proteins with Val and Thr at P1' could not undergo Met cleavage. These in vitro data were fully consistent with data for 862 E. coli proteins with known N-terminal sequences in vivo. The specificity sites were found to be identical to those for the other type of MAPs, MAP2s, and a dedicated prediction tool for Met cleavage is now available. Taking into account the rules of MAP cleavage and leader peptide removal, the N termini of all proteins were predicted from the annotated genome and compared with data obtained in vivo. This analysis showed that proteins displaying N-Met cleavage are overrepresented in vivo. We conclude that protein secretion involving leader peptide cleavage is more frequent than generally thought.