Relationship between conserved consensus site residues and the productive conformation for the TPQ cofactor in a copper-containing amine oxidase from yeast

A highly conserved asparagine residue is contained in the consensus site sequences of all known copper-containing amine oxidases (CAOs). On the basis of published crystallographic structures, the asparagine is found to reside proximal to the active site redox cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ). In this study, the conserved asparagine was changed to an alanine in a CAO from Hansenula polymorpha expressed in Saccharomyces cerevisiae, and the mutant's catalytic properties were characterized using steady-state kinetics and resonance Raman spectroscopy. Several lines of evidence point to TPQ exisiting in an nonproductive orientation in the mutant, including reductions in several steady-state parameters and an accumulation of an inactive product Schiff base complex when the enzyme is incubated with methylamine as the substrate. This product Schiff base complex was previously found to form following mutation of another conserved consensus site residue, a glutamate (or aspartate) at the C + 1 position from TPQ [Cai, D., Dove, J., Nakamura, N., Sanders-Loehr, J., and Klinman, J. P. (1997) Biochemistry 36, 11472-11478]. The results suggest that these two residues are crucial in maintaining the balance of cofactor mobility versus rigidity expected to be necessary during the dual processes of biogenesis and catalysis, respectively, that all CAOs must accomplish. In addition, a previously unidentified structural linkage between these two highly conserved residues is proposed which spans both subunits of the dimeric CAOs, and may have implications for intersubunit communication.     

Characterization of recombinant Saccharomyces cerevisiae manganese-containing superoxide dismutase and its H30A and K170R mutants expressed in Escherichia coli

All known Mn-containing superoxide dismutases (MnSODs) have a highly conserved histidine (His-30 in Escherichia coli FeSOD) in the active-site channel, and nearly all have an active-site arginine (Arg-170) that has been proposed to play a combined structural and functional role [Chan et al., Arch. Biochem. Biophys. 279, 195-201 (1990)]. In Saccharomyces cerevisiae MnSOD, the active-site arginine is replaced by a lysine. The S. cerevisiae MnSOD gene has been cloned and expressed in E. coli, and H30A and K170R site-specific mutants have been prepared. The purified recombinant native (RN) and mutant enzymes were compared to one another and to the native enzyme purified from S. cerevisiae (SC) in terms of activity, temperature stability, and sensitivity to 2,4,6-trinitrobenzenesulfonate (TNBS) and phenylglyoxal (PG). All enzymes had high specific activities (SC = 5000, RN = 5600, H30A = 4500, K170R = 4600) (U/mg, using the pyrogallol assay). SC, RN, and H30A were very stable at 75 degreesC (pH 8.0), with half-lives of 4.7, 2.8, and 2.7 h, respectively, while K170R had a much greater temperature lability, with a half-life of 0.36 h under these conditions. TNBS (0.5 mM, pH 9.0, 25 degreesC) rapidly inactivated SC, RN, and H30A, with half-lives of 3. 5, 5.1, and 5.5 min, respectively, but only slowly inactivated K170R, with a half-life of 101 min. PG (20 mM, pH 9.0, 25 degreesC) caused very slow inactivation of SC, RN, and H30A by biphasic kinetics, and each enzyme retained >/=25% activity after 3 h of modification. K170R, on the other hand, was completely inactivated by PG under these conditions by first-order kinetics, with a half-life of 7.0 min. The data suggest that His-30, a residue highly conserved in the active-site channel of MnSODs and FeSODs, does not play a crucial role in catalysis or stability. In addition, Lys-170, a residue that is almost always arginine in the numerous other MnSODs and FeSODs sequenced to date, can be replaced by arginine with no loss of catalytic activity, but K170R is less stable and Arg-170 in this mutant is more exposed than the corresponding arginine in other SODs. RN and SC showed some surprising differences. Thus, while the specific activities of RN and SC are very similar, SC is more stable to inactivation at 75 degreesC, and less susceptible to inactivation by phenylglyoxal, than RN. These data suggest that there may be slight differences in the tertiary structures of SC, the native enzyme expressed in S. cerevisiae, and RN, the recombinant native enzyme expressed in E. coli.     

The critical active-site amine of the human 8-oxoguanine DNA glycosylase, hOgg1: direct identification, ablation and chemical reconstitution

BACKGROUND: Base-excision DNA repair (BER) is the principal pathway responsible for the removal of aberrant, genotoxic bases from the genome and restoration of the original sequence. Key components of the BER pathway are DNA glycosylases, enzymes that recognize aberrant bases in the genome and catalyze their expulsion. One major class of such enzymes, glycosylase/lyases, also catalyze scission of the DNA backbone following base expulsion. Recent studies indicate that the glycosylase and lyase functions of these enzymes are mechanistically unified through a common amine-bearing residue on the enzyme, which acts as both the electrophile that displaces the aberrant base and an electron sink that facilitates DNA strand scission through imine (Schiff base)/conjugate elimination chemistry. The identity of this critical amine-bearing residue has not been rigorously established for any member of a superfamily of BER glycosylase/lyases. RESULTS: Here, we report the identification of the active-site amine of the human 8-oxoguanine DNA glycosylase (hOgg1), a human BER superfamily protein that repairs the mutagenic 8-oxoguanine lesion in DNA. We employed Edman sequencing of an active-site peptide irreversibly linked to substrate DNA to identify directly the active-site amine of hOgg1 as the epsilon-NH2 group of Lys249. In addition, we observed that the repair-inactive but recognition-competent Cys249 mutant (Lys249-->Cys) of hOgg1 can be functionally rescued by alkylation with 2-bromoethylamine, which functionally replaces the lysine residue by generating a gamma-thia-lysine. CONCLUSIONS: This study provides the first direct identification of the active-site amine for any DNA glycosylase/lyase belonging to the BER superfamily, members of which are characterized by the presence of a helix-hairpin-helix-Gly/Pro-Asp active-site motif. The critical lysine residue identified here is conserved in all members of the BER superfamily that exhibit robust glycosylase/lyase activity. The ability to trigger the catalytic activity of the Lys249-->Cys mutant of hOgg1 by treatment with the chemical inducer 2-bromoethylamine may permit snapshots to be taken of the enzyme acting on its substrate and could represent a novel strategy for conditional activation of catalysis by hOgg1 in cells.     

Irreversible inactivation of protein kinase C by a peptide-substrate analog

Protein kinase C (PKC) is a phospholipid-dependent isozyme family that plays a pivotal role in mammalian signal-transduction pathways that mediate cell growth and differentiation and pathological developments, such as the acquisition of drug resistance by cancer cells. Several peptide-substrate analogs have been shown to reversibly inhibit PKC with high potency and selectivity, but peptide-substrate analogs that antagonize PKC by forming a covalent complex with the enzyme have not been reported. The development of active site-directed irreversible inactivators of PKC could provide new insights into the catalytic mechanism and might ultimately lead to the design of novel therapeutics targeted at PKC. In this report, we show that the peptide-substrate analog Arg-Lys-Arg-Cys-Leu-Arg-Arg-Leu (RKRCLRRL) irreversibly inactivates PKC in a dithiothreitol-sensitive manner. The inactivation mechanism most consistent with our results is the formation of a covalent linkage between the inhibitor-peptide and the enzyme at its active-site. Limited proteolysis of PKC produces a catalytic-domain fragment that is independent of the phospholipid cofactor. RKRCLRRL antagonized the histone kinase activity of PKC and its catalytic-domain fragment with similar efficacies, achieving > 50% inactivation at an RKRCLRRL concentration of 10 microM. In contrast, RKRCLRRL analogs with single amino acid substitutions at Cys were non-inhibitory. The inactivated complex of the catalytic-domain fragment and RKRCLRRL was stable upon dilution, and the inactivation of PKC and the catalytic-domain fragment by RKRCLRRL was quenched by dithiothreitol, providing evidence that the enzyme and the synthetic peptide may be covalently linked in an inactivated complex by a disulfide bond. Substrates and substrate analogs protected the catalytic-domain fragment against inactivation by RKRCLRRL, providing evidence that inactivation entailed binding of RKRCLRRL at the active-site of the enzyme. S-Thiolation is the formation of mixed disulfides between proteins and low molecular weight thiols. PKC is thought to have a highly reactive Cys residue in its active-site, and Cys residues that are flanked by basic residues, as is the case in RKRCLRRL, display enhanced reactivity. Our results support an inactivation mechanism that entails S-thiolation of the active-site of PKC by RKRCLRRL. This is the first report of irreversible inactivation of PKC by an active site-directed peptide.     

Paracatalytic self-inactivation of fructose-1,6-bisphosphate aldolase. Structure of the crosslink formed at the active site

Oxidation of enzyme-substrate carbanion intermediates by extrinsic oxidants may result in irreversible paracatalytic inactivation of certain enzymes. In paracatalytically modified fructose-1,6-bisphosphate aldolase from rabbit muscle the polypeptide chain had been found to be crosslinked at active-site Lys229 (Schiff base forming with substrate) and Lys146 by a phosphorylated three-carbon moiety [Lubini, D. G. E. and Christen, P. (1979) Proc. Natl Acad. Sci. USA 76, 2527-2531]. In the present study, the structure of this crosslink was elucidated by instrumental analysis. Aldolase was paracatalytically modified in the presence of fructose 1,6-bisphosphate and hexacyanoferrate(III). The completely inactivated enzyme was digested with pronase. The crosslinked peptide was isolated by gel filtration and reverse-phase HPLC. Mass spectroscopy, 1H- and 13C-NMR showed that a derivative of dihydroxyacetone phosphate forms an amidine with the epsilon-amino groups of the two lysine residues: [formula: see text]     

Role of lysine 39 of alanine racemase from Bacillus stearothermophilus that binds pyridoxal 5'-phosphate. Chemical rescue studies of Lys39 --> Ala mutant

The lysine residue binding with the cofactor pyridoxal 5'-phosphate (PLP) plays an important role in catalysis, such as in the transaldimination and abstraction of alpha-hydrogen from a substrate amino acid in PLP-dependent enzymes. We studied the role of Lys39 of alanine racemase (EC 5.1.1.1) from Bacillus stearothermophilus, the PLP-binding residue of the enzyme, by replacing it site-specifically with alanine and characterizing the resultant K39A mutant enzyme. The mutant enzyme turned out to be inherently inactive, but gained an activity as high as about 0.1% of that of the wild-type enzyme upon addition of 0.2 M methylamine. The amine-assisted activity of the mutant enzyme depended on the pKa values and molecular volumes of the alkylamines used. A strong kinetic isotope effect was observed when alpha-deuterated D-alanine was used as a substrate in the methylamine-assisted reaction, but little effect was observed using its antipode. In marked contrast, only L-enantiomer of alanine showed a solvent isotope effect in deuterium oxide in the methylamine-assisted reaction. These results suggest that methylamine serves as a base not only to abstract the alpha-hydrogen from D-alanine but also to transfer a proton from water to the alpha-position of the deprotonated (achiral) intermediate to form D-alanine. Therefore, the exogenous amine can be regarded as a functional group fully representing Lys39 of the wild-type enzyme. Lys39 of the wild-type enzyme probably acts as the base catalyst specific to the D-enantiomer of alanine. Another residue specific to the L-enantiomer in the wild-type enzyme is kept intact in the K39A mutant.     

Active site labeling of erythrocyte transglutaminase by o-phthalaldehyde

Tissue-type transglutaminase is inactivated in a time-dependent way during incubation with submillimolar concentrations of o-phthalaldehyde, with affinity labeling kinetics. The rate of inactivation by the reagent is greatly enhanced in the presence of the essential enzyme cofactor calcium and is decreased by GTP, an allosteric inhibitor. A fluorescent isoindole derivative is formed during the modification apparently through crosslinkage of active site Cys 277 to a lysine residue. These data and the quenching of fluorescence by addition of calcium ions suggest that the enzyme active site is directly involved in the inactivation process.     

p53 protein expression in breast cancer as related to histopathological characteristics and prognosis

Reaction of Klebsiella aerogenes urease with diethylpyrocarbonate (DEP) led to a pseudo-first-order loss of enzyme activity by a reaction that exhibited saturation kinetics. The rate of urease inactivation by DEP decreased in the presence of active site ligands (urea, phosphate, and boric acid), consistent with the essential reactive residue being located proximal to the catalytic center. The pH dependence for the rate of inactivation indicated that the reactive residue possessed a pKa of 6.5, identical to that of a group that must be deprotonated for catalysis. Full activity was restored when the inactivated enzyme was treated with hydroxylamine, compatible with histidinyl or tyrosinyl reactivity. Spectrophotometric studies were consistent with DEP derivatization of 12 mol of histidine/mol of native enzyme. In the presence of active site ligands, however, approximately 4 mol of histidine/mol of protein were protected from reaction. Each protein molecule is known to possess two catalytic units; hence, we propose that urease possesses at least one essential histidine per catalytic unit.     

Structure of the complex between pyridoxal 5'-phosphate and the tyrosine 225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase determined by isotope-edited classical Raman difference spectroscopy

The azomethine (Schiff base) linkage between the epsilon-amino group of active-site lysine 258 and the carbonyl moiety of enzyme-bound pyridoxal 5'-phosphate (PLP) normally exhibits absorbance maxima at ca. 360 (high-pH form) or ca. 430 nm (low-pH form). However, the absorbance maximum is shifted from 358 to 386 nm, a value which is similar to that of free PLP (lambda max = 388 nm), in a mutant form of Escherichia coli aspartate aminotransferase (AATase) in which tyrosine 225, which normally donates a hydrogen bond to the phenolate function of PLP, has been replaced with phenylalanine (Y225F). This spectral shift suggested that PLP binds to Y225F as the free aldehyde. The following evidence from isotope-edited classical Raman spectroscopy proves conclusively that the near-UV spectrum is anomalous and that PLP is bound to Y225F as a Schiff base: (1) A strong cofactor peak at 1630 cm-1 in the holoenzyme-minus-apoenzyme difference spectrum of the unprotonated form of Y225F is red-shifted by 18 cm-1 in enzyme labeled with 15N at lysine 258 and other positions. (2) This isotope-induced red shift is similar to that observed in the unprotonated form of the model Schiff base, PLP-valine. (3) The Raman spectrum of Y225F is unchanged in H(2)18O, while peaks at ca. 1670 cm-1 in the spectrum of free PLP or in that of a mutant of AATase in which Lys-258 is replaced with Ala, are red-shifted by ca. 30 cm-1 in H(2)18O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalanines

beta,beta-Dichloro- and beta,beta,beta-trifluoroalanine irreversibly inactivate a number of pyridoxal phosphate dependent enzymes which catalyze beta- or gamma-elimination reactions. The inactivation is time dependent and the rate of inactivation is first order in enzyme concentration. This suggests that inactivation is due to covalent modification of the enzyme by a species generated at the active site from the polyhaloalanine (i.e., suicide inactivation). Monohaloalanines are substrates and do not inactivate. For gamma-cystathionase, covalent and stoichiometric attachment of [1-14C]beta,beta,beta-trifluoroalanine was shown. It is proposed that the mechanism of inactivation involves Schiff base formation between inactivator and enzyme-bound pyridoxal and subsequent elimination of HC1 from dichloroalanine or HF from trifluoroalanine. This results in the formation of a beta-halo-alpha,beta unsaturated imine, an activated Michael acceptor. Michael addition of a nucleophile at the active site leads to covalent labeling of the enzyme and inactivation. Alanine racemase is also inactivated by the two polyhaloalanines. Glutamate-pyruvate and gultamate-oxaloacetate transaminase are inactivated by monohaloalanines but not by polyhaloalanines.     

Mutational analysis of potential zinc-binding residues in the active site of the enterococcal D-Ala-D-Ala dipeptidase VanX

VanX, one of the five proteins required for the vancomycin-resistant phenotype in clinically pathogenic Enterococci, is a zinc-containing d-Ala-d-Ala dipeptidase. To identify potential zinc ligands and begin defining the active site residues, we have mutated the 2 cysteine, 5 histidine, and 4 of the 28 aspartate and glutamate residues in the 202 residue VanX protein. Of 10 mutations, 3 cause inactivation and greater than 90% loss of zinc in purified enzyme samples, implicating His116, Asp123, and His184 as zinc-coordinating residues. Homology searches using the 10 amino acid sequence SxHxxGxAxD, in which histidine and aspartate residues are putative zinc ligands, identified the metal coordinating ligands in the N-terminal domain of the murine Sonic hedgehog protein, which also exhibits an architecture for metal coordination identical to that observed in thermolysin from Bacillus thermoproteolyticus. Furthermore, this 10 amino acid consensus sequence is found in the Streptomyces albus G zinc-dependent N-acyl-d-Ala-d-Ala carboxypeptidase, an enzyme catalyzing essentially the same d-Ala-d-Ala dipeptide bond cleavage as VanX, suggesting equivalent mechanisms and zinc catalytic site architectures. VanX residue Glu181 is analogous to the Glu143 catalytic base in B. thermoproteolyticus thermolysin, and the E181A VanX mutant has no detectable dipeptidase activity, yet maintains near-stoichiometric zinc content, a result consistent with the participation of the residue as a catalytic base.     

Serologic diagnosis of canine and equine borreliosis: use of recombinant antigens in enzyme-linked immunosorbent assays

A key regulatory enzyme of the C4-photosynthetic pathway is stromal pyruvate,orthophosphate dikinase (PPDK, EC 2.7.9.1). As a pivotal enzyme in the C4 pathway, it undergoes diurnal light-dark regulation of activity which is mediated by a single bifunctional regulatory protein (RP). RP specifically inactivates PPDK in the dark by an ADP-dependent phosphorylation of an active-site Thr residue (Thr-456 in maize). Conversely, RP activates inactive PPDK in the light by phosphorolytic dephosphorylation of this target Thr-P residue. We have employed a His-tagged maize recombinant C4 PPDK for directed mutagenesis of this active-site regulatory Thr. Three such mutants (T456V, T456S, T456D) were analyzed with respect to overall catalysis and regulation by exogenous maize RP. Substitution with Val and Ser at this position does not affect overall catalysis, whereas Asp abolishes enzyme activity. With respect to regulation by RP, it was found that Ser can effectively substitute for the wild-type Thr residue in that mutant enzyme is phosphorylated and inactivated by RP. The T456V mutant, however, could not be phosphorylated and was, thus, resistant to ADP-dependent inactivation by RP.     

Inactivation of 4-oxalocrotonate tautomerase by 2-oxo-3-pentynoate

The compound, 2-oxo-3-pentynoate, has been synthesized and tested as an inhibitor of the enzyme 4-oxalocrotonate tautomerase. The enzyme is rapidly and irreversibly inactivated by the acetylenic product analogue in a time-dependent fashion. The enzyme displays saturation kinetics and is protected from inactivation by the presence of substrate. These observations are consistent with inactivation taking place at the active site. Partial reactivation ( approximately 18%) occurs by incubating the inactivated enzyme with 10 mM hydroxylamine (pH 7.3). The partition ratio, determined to be approximately 0.4, suggests that the inactivation of 4-OT by 2-oxo-3-pentynoate shows half-of-the-sites stoichiometry. The same phenomenon is observed in the inactivation of 4-OT by 3-bromopyruvate and can be explained by examination of the crystal structure. Mass spectral analysis shows that a single residue is modified on the enzyme which has been localized to the nine residue amino-terminal fragment Pro-1 to Glu-9. It can be reasonably concluded that Pro-1 is the site of covalent attachment. Inactivation of 4-OT can occur by either a Michael addition of 4-OT to C-4 of 2-oxo-3-pentynoate or by the enzyme-catalyzed rearrangement of 2-oxo-3-pentynoate to an allene derivative which alkylates Pro-1. These results provide the foundation for the use of 2-oxo-3-pentynoate in future mechanistic studies and as a ligand in an inactivated 4-OT complex that can be studied by X-ray crystallography. Finally, 2-oxo-3-pentynoate is an acetylene analogue of a variety of 2-oxo acids and as such may have general utility as an inhibitor of reactions that bind and process these compounds.     

Exploring a channel to the active site of copper/topaquinone-containing phenylethylamine oxidase by chemical modification and site-specific mutagenesis

Copper amine oxidase contains an organic redox cofactor, 2,4, 5-trihydroxyphenylalaninequinone (topaquinone, TPQ), derived by the post-translational modification of a specific tyrosyl residue. To identify amino acid residues participating in the biogenesis of TPQ in the recombinant phenylethylamine oxidase from Arthrobacter globiformis, we have modified the copper/TPQ-less apoenzyme and the copper/TPQ-containing holoenzyme with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-F). In the apoenzyme modification, the Cu2+-dependent, self-processing formation of the TPQ cofactor was retarded in accordance with the amount of NBD incorporated. The holoenzyme was also rapidly inactivated by incubation with NBD-F. The inactivation was prevented almost completely in the presence of an oxidation product from phenylethylamine, phenylacetaldehyde. Furthermore, the reaction of an inhibitor, phenylhydrazine, with TPQ was much slower in the NBD-labeled holoenzyme than in the native holoenzyme. Sequence analysis of the NBD-labeled holoenzyme has identified Lys184 and Lys354 as the labeled sites. The two Lys residues are located close to the entrance to a channel, which has been found by recent X-ray crystallographic studies to be suitable for the movement of substrates and products to and from the Cu2+/TPQ-active site buried in the protein interior (Wilce, M. C. J., et al. (1997) Biochemistry 36, 16116-16133). However, site-specific mutant enzymes for Lys184, Lys354, and the neighboring invariant His355 had normal capacities for the TPQ formation in apoenzyme. These residues were also found to be dispensable for catalytic activity of holoenzyme. Thus, modification of Lys184 and Lys354 with NBD-F presumably causes structural perturbations of the substrate channel or steric hindrance for the access of small molecules to the active site through the channel.     

Modulation of the catalytic activity of brain succinic semialdehyde reductase by reaction with pyridoxal 5'-phosphate

An NADPH-dependent succinic semialdehyde reductase from bovine brain was inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After sodium borohydride reduction of the inactivated enzyme, it was observed that 1 mol phosphopyridoxyl residue was incorporated/mol enzyme monomer. The coenzyme, NADPH, protected the enzyme against inactivation by pyridoxal 5'-phosphate. After tryptic digestion of the enzyme modified with pyridoxal 5'-phosphate in the presence and absence of NADPH followed by [1H]NaBH4 reduction, a radioactive peptide absorbing at 310 nm was isolated by reverse-phase HPLC. The amino acid sequence of the peptide identified a portion of the pyridoxal-5'-phosphate-binding site as the region containing the sequence I-L-E-N-I-Q-V-F-X-K, where X indicates that the phenylthiohydantoin amino acid could not be assigned. The missing residue, however, can be designated as a phosphopyridoxyl lysine as interpreted from the result of amino acid composition of the peptide. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of pyridoxal 5'-phosphate to a specific lysyl residue at or near the coenzyme-binding site of the protein.     

Identification of Asp258 as the metal coordinate of pigeon liver malic enzyme by site-specific mutagenesis

Pigeon liver malic enzyme was inactivated by ferrous sulfate in the presence of ascorbate. Manganese and some other divalent metal ions provided complete protection of the enzyme against the Fe(2+)-induced inactivation. The inactivated enzyme was subsequently cleaved by the Fe(2+)-ascorbate system at Asp258-Ile259, which was presumably the Mn(2+)-binding site of the enzyme [Wei, C. H., Chou, W. Y., Huang, S. M., Lin, C. C., & Chang, G. G. (1994) Biochemistry 33, 7793-7936]. For identification of Asp258 as the putative metal-binding site of the enzyme, we prepared four mutant enzymes substituted at Asp258 with glutamate (D258E), asparagine (D258N), lysine (D258K), or alanine (D258A), respectively. These mutant proteins were recombinantly expressed in a bacterial expression system (pET-15b) with a stretch of histidine residues attached at the N-terminus and were successfully purified to apparent homogeneity by a single Ni-chelated affinity column. Among the four mutants, only D258E possessed 0.8% residual activity after purification; all other purified mutants had < 0.0001% residual activity in catalyzing the oxidative decarboxylation of L-malate. The D258E mutant was susceptible to inactivation by the Fe(2+)-ascorbate system, albeit with much slower inactivation rate, and was protected by the Mn2+ to a lesser extent as compared to the wild-type enzyme. None of the mutants were cleaved by the Fe(2+)-ascorbate system under conditions that cleaved the natural or wild-type enzyme at Asp258.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase

o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme.     

Histidine decarboxylase of Lactobacillus 30a: inactivation and active-site labeling by L-histidine methyl ester

Histidine decarboxylase from Lactobacillus 30a is rapidly and irreversibly inactivated upon incubation with L-histidine methyl ester. The rate of inactivation is first-order with respect to remaining active enzyme and exhibits saturation kinetics with a kinact of 1.2 mM and an apparent first-order rate constant of 0.346 min-1 at pH 4.8 and 25 degrees C. On complete inactivation, 3 mol of [14C]histidine (from L-[14C]histidine methyl ester) and 2 mol of 14C (from L-histidine [14C]methyl ester) are bound in nondialyzable form per mol (190 000 g) of protein inactivated with a corresponding loss of three of the five DTNB-titratable--SH groups that are essential for activity of the native enzyme. Imidazole propionate, a competitive inhibitor of the enzyme, protects against inactivation, loss of --SH groups, and incorporation of radioactivity from both the histidine and the methyl ester moieties of the labeled inhibitor, and kinetic evidence indicates that imidazole propionate and histidine methyl ester compete for binding at the active site of histidine decarboxylase in a mutually exclusive manner. Treatment of the labeled protein with either alkali or hydroxylamine results in the quantitative release of radioactivity. These data suggest that inactivation of histidine decarboxylase by L-histidine methyl ester results from two different modes of interaction between the inhibitor and the active site of histidine decarboxylase; the major interaction involves an essential -SH group.     

Oxidation of phenolic compounds by lactoperoxidase. Evidence for the presence of a low-potential compound II during catalytic turnover

The lactoperoxidase (LPO)-catalyzed oxidation of p-phenols by hydrogen peroxide has been studied. The behavior of the enzyme differs from that of other peroxidases in this reaction. In particular LPO shows several catalytic intermediates during the catalytic cycle because of its capability to delocalize an oxidizing equivalent on a protein amino acid residue. In the phenol oxidation the enzyme Compound I species, containing an iron-oxo and a protein radical, uses the iron-oxo group at acidic pH and the protein radical in neutral or basic medium. Kinetic and spectroscopic studies indicate that the ionization state of an amino acid residue with pKa 5.8 +/- 0.2, probably the distal histidine, controls the enzyme intermediate forms at different pH. LPO undergoes inactivation during the oxidation of phenols. The inactivation is reversible and depends on the easy formation of Compound III even at low oxidant concentration. The inactivation is due to the substrate redox potential since the best substrate is that with lowest redox potential, while the worst substrate has the highest potential. This strongly indicates that Compound II, formed during catalytic turnover, has a low redox potential, making easier its oxidation by hydrogen peroxide to Compound III. The dependence of LPO activity on the phenols redox potential suggests that the protein radical where an oxidizing equivalent can be localized is a tyrosyl residue.     

The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology

BACKGROUND: Purine nucleoside phosphorylase (PNP) from Escherichia coli is a hexameric enzyme that catalyzes the reversible phosphorolysis of 6-amino and 6-oxopurine (2'-deoxy)ribonucleosides to the free base and (2'-deoxy)ribose-1-phosphate. In contrast, human and bovine PNPs are trimeric and accept only 6-oxopurine nucleosides as substrates. The difference in the specificities of these two enzymes has been utilized in gene therapy treatments in which certain prodrugs are cleaved by E. coli PNP but not the human enzyme. The trimeric and hexameric PNPs show no similarity in amino acid sequence, even though they catalyze the same basic chemical reaction. Structural comparison of the active sites of mammalian and E. coli PNPs would provide an improved basis for the design of potential prodrugs that are specific for E. coli PNP. RESULTS: The crystal structure of E. coli PNP at 2.0 A resolution shows that the overall subunit topology and active-site location within the subunit are similar to those of the subunits from human PNP and E. coli uridine phosphorylase. Nevertheless, even though the overall geometry of the E. coli PNP active site is similar to human PNP, the active-site residues and subunit interactions are strikingly different. In E. coli PNP, the purine- and ribose-binding sites are generally hydrophobic, although a histidine residue from an adjacent subunit probably forms a hydrogen bond with a hydroxyl group of the sugar. The phosphate-binding site probably consists of two main-chain nitrogen atoms and three arginine residues. In addition, the active site in hexameric PNP is much more accessible than in trimeric PNP. CONCLUSIONS: The structures of human and E. coli PNP define two possible classes of nucleoside phosphorylase, and help to explain the differences in specificity and efficiency between trimeric and hexameric PNPs. This structural data may be useful in designing prodrugs that can be activated by E. coli PNP but not the human enzyme.