Photochemical inactivation of <i>Aeromonas</i> aminopeptidase

Mäkinen, Kauko K.

doi:10.1016/0014-5793(81)80911-8

Cited by 7 publications

(4 citation statements)

References 12 publications

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“…PslB (3.2 mg/ml) was incubated with different concentrations of 2,3‐butanedione (150 mM borate buffer, pH 9.0) at 22 °C in the dark for 0–60 min [12,13], then the excess 2,3‐butanedione was removed by passing the reaction mixture through a Sephadex G‐25 column (Pharmacia). The PMI activity was measured as described above.…”

Section: Methodsmentioning

confidence: 99%

Identification of amino acid residues important for the phosphomannose isomerase activity of PslB in Pseudomonas aeruginosa PAO1

et al. 2008

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Phosphomannose isomerase (PMI) plays a pivotal role in biosynthesis of GDP-mannose, an important precursor of many polysaccharides. We demonstrate in this study that Pseudomonas aeruginosa pslB encodes a protein with GDP-mannose pyrophosphorylase/PMI dual activities. The PMI activity is Co 2+ -dependent and could be inhibited by GDP-mannose in a competitive manner. Furthermore, the activity could be inactivated by 2,3-butanedione suggesting the presence of a catalytic Arg residue. Site-specific mutations at R373, R472, R479, E410, H411, N433 and E458 increase the K M approximately 8-20-fold. The PMI activity of PslB was completely diminished with a R408K or R408A, reflecting the importance of this residue in catalysis. Overall, these results provide a basis for understanding the catalytic mechanism of PMI.

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Section: Methodsmentioning

confidence: 99%

Identification of amino acid residues important for the phosphomannose isomerase activity of PslB in Pseudomonas aeruginosa PAO1

et al. 2008

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“…The purpose of these studies was to elucidate the nature of the active amino acid residues which possibly are involved in GGT activity by limiting the tests to include typical, widely employed chemical modification reactions and enzyme affectors used under standard conditions described in the literature. Consequently, modification with 2,3-butanedione and phenylglyoxal (to study the possible involvement of active arginyl residues) was carried out under protection from light (25). Diethylpyrocarbonate (which normally reacts with histidyl or tyrosyl residues) (30), diisopropylfluorophosphate (DFP) (which is known to inhibit serine enzymes), tetranitromethane (tested primarily for tyrosyl residues) (36,43), and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ; highly specific for active carboxyl groups) (35,38), were studied as described in cited references.…”

Section: Methodsmentioning

confidence: 99%

gamma-Glutamyltransferase from the outer cell envelope of Treponema denticola ATCC 35405

Mäkinen

1997

Infect Immun

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The human oral spirochete Treponema denticola ATCC 35405 was shown to exhibit relatively high enzyme activity toward the ␥-glutamyl amide bond present in N-␥-L-glutamyl-4-nitroaniline. The enzyme responsible for this catalysis (␥-glutamyltransferase [GGT]; EC 2.3.2.2) was purified by means of fast protein liquid chromatography to two sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-pure forms from a mild (0.1%) Triton X-100 extract of washed cells. The GGT was studied primarily with regard to its hydrolytic activity by using N-␥-L-glutamyl-4-nitroaniline as a substrate, although the GGT was shown to catalyze transpeptidation reactions. The high-molecular-mass form of the GGT gave a value of about 213 kDa by SDS-PAGE when heat treatment was omitted and one of 26 kDa after heat treatment; mass spectrometry gave a value of 26.877. The larger form may represent an aggregate with nonprotein structures (possibly of a carbohydrate nature). The preliminary N-terminal sequence of the GGT is MKKPLIGITGSXLYETSQXXF. The enzyme was highly active on glutathione, transferring its Glu residue either to a water molecule or to the Gly-L-Leu dipeptide. The GGT stability was absolutely dependent on the presence of free thiol(s), while no evidence of metalloenzyme nature was obtained. The proposed location of the GGT in the outer cell envelope and its high activity on glutathione, a major nonprotein thiol present in virtually all cells, suggest that the GGT may play a role in the propagation of T. denticola within inflamed periodontal tissues.

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“…Thus these resuits suggest that EEDQ has reacted with a reactive residue that is essential to the activity of the enzyme and that reaction of one mole of EEDQ per active site of benzoylarginine peptidase had probably caused this total enzyme inaotivation. Collectively, these data showed that the inactivation of the enzyme by EEDQ was time-, concentration-, and pH-dependent, in previous studies (Pougeois ef ai, 1978;Saccomani ef at., 1981;Phelps and Hafeti, 1984;Makinen and Makinen, 1987;Makinen et at., 1982) such kinetic behaviour has been interpreted in terms of EEDQ reacting with an active carboxyl residue. The presence of BAPNA or BANA protected against the EEDQ-induced inactivation by 50-85% (both substrates were used at 5-25mM concentrations).…”

Section: Effect Of Woodward's Reagent K (Wrk)mentioning

confidence: 99%

The benzoylarginine peptidase from Treponema denticola (strain ASLM), a human oral spirochaete: evidence for active‐site carboxyl groups

Mäkinen

Chen

Mäkinen

et al. 1990

Molecular Microbiology

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The benzoylarginine peptidase of Treponema denticola (strain ASLM; a human oral spirochaete) was progressively and irreversibly inactivated by 1-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline, a carboxyl-group reagent. At acidic pH values, reaction of one mole of the modifier per active site of the enzyme resulted in total inactivation of the enzyme. Assuming that this modifier is a specific carboxyl reagent, the data suggest that the inactivation of the T. denticola benzoylarginine peptidase was caused by the modification of one carboxyl group located close to the active site of the enzyme. Results obtained with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulphonate) supported these findings. Carbethoxylation with diethylpyrocarbonate effectively inactivated the enzyme, and addition of hydroxylamine at pH 7.0 restored the activity almost totally, suggesting that the pyrocarbonate had reacted with tyrosyl or histidyl residues.

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Photochemical inactivation of Aeromonas aminopeptidase

Cited by 7 publications

References 12 publications

Identification of amino acid residues important for the phosphomannose isomerase activity of PslB in Pseudomonas aeruginosa PAO1

Identification of amino acid residues important for the phosphomannose isomerase activity of PslB in Pseudomonas aeruginosa PAO1

gamma-Glutamyltransferase from the outer cell envelope of Treponema denticola ATCC 35405

The benzoylarginine peptidase from Treponema denticola (strain ASLM), a human oral spirochaete: evidence for active‐site carboxyl groups

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