gamma-Glutamyltransferase from the outer cell envelope of Treponema denticola ATCC 35405.
The human oral spirochete Treponema denticola ATCC 35405 was shown to exhibit relatively high enzyme activity toward the gamma-glutamyl amide bond present in N-gamma-L-glutamyl-4-nitroaniline. The enzyme responsible for this catalysis (gamma-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-gamma-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.
Mäkinen
P L
PL
Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor 48109, USA.
Mäkinen
K K
KK
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D002614:Chelating Agents / Q000494:pharmacology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002712:Chlorides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014166:Transferases / Q000378:metabolism; D014208:Treponema / Q000737:chemistry / Q000201:enzymology*; D005723:gamma-Glutamyltransferase / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification
D001425:Bacterial Outer Membrane Proteins; D002614:Chelating Agents; D002712:Chlorides; D007202:Indicators and Reagents; D013438:Sulfhydryl Compounds; D014166:Transferases; D005723:gamma-Glutamyltransferase