The leucyl aminopeptidase from Helicobacter pylori is an allosteric enzyme.
This study describes the cloning, genetic analysis and biochemical characterization of a leucyl aminopeptidase (LAP) from Helicobacter pylori. A gene encoding LAP was cloned from H. pylori and the expressed 55 kDa protein displayed homology to aminopeptidases from Gram-negative bacteria, plants and mammals. This LAP demonstrated amidolytic activity against L-leucine-p-nitroanilide. Optimal activity was observed at pH 8.0 and 45 degrees C, with V(max) of 232 mumol min(-1) (mg protein)(-1) and S(0.5) of 0.65 mM. The data suggest that LAP could be allosteric (n(H)=2.27), with regulatory homohexamers, and its activity was inhibited by ion chelators and enhanced by divalent manganese, cobalt and nickel cations. Bestatin inhibited both LAP activity (IC(50)=49.9 nM) and H. pylori growth in vitro. The results point to the potential use of LAP as a drug target to develop novel anti-H. pylori agents.
Dong
Lei
L
State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China.
Cheng
Ni
N
State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China.
Wang
Ming-Wei
MW
The National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P. R. China.
Zhang
Junfeng
J
State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China.
Shu
Chang
C
State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China.
Zhu
De-Xu
DX
State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China.
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000813:Anilides / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D003067:Coenzymes; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D016480:Helicobacter pylori / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007931:Leucyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D000813:Anilides; D002413:Cations, Divalent; D002614:Chelating Agents; D003067:Coenzymes; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; C001536:1-leucine-4-nitroanilide; D007931:Leucyl Aminopeptidase; D007930:Leucine; C012211:ubenimex