Arg(362) and Tyr(365) of the botulinum neurotoxin type a light chain are involved in transition state stabilization.
The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn(2+) is coordinated by His(222) and His(226) of the Zn(2+) binding motif HisGluXXHis and Glu(261), whereas Glu(223) coordinates the water molecule required for hydrolysis as the fourth ligand. Recent analysis of a cocrystal of the BoNT/B LC and its substrate synaptobrevin 2 suggested that Arg(362) and Tyr(365) of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu(350) which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg(362) and substitution of Tyr(365) with Phe resulted in 79- and 34-fold lower k(cat)/K(m) values, respectively. These changes were provoked by decreased catalytic rates (k(cat)) and not by alterations of ground state substrate binding as evidenced by largely unchanged K(d) and K(m) values. None of these mutations affected the overall secondary structure or zinc content of the LC. These findings suggest that the guanidino group of Arg(362) and the hydroxyl group of Tyr(365) together accomplish transition state stabilization as was proposed for thermolysin, being the prototypical member of the gluzincin superfamily of metalloproteases. Mutation of Glu(350) dramatically diminished the hydrolytic activity which must partly be attributed to an altered active site fine structure as demonstrated by an increased sensitivity toward heat-induced denaturing and a lower Zn(2+) binding affinity. Glu(350) apparently occupies a central position in the active site and presumably positions His(222) and Arg(362).
Binz
Thomas
T
Department of Biochemistry and Department of Biophysical Chemistry, Medizinische Hochschule Hannover, Hannover, Germany. binz.thomas@mh-hannover.de
Bade
Steffen
S
Rummel
Andreas
A
Kollewe
Astrid
A
Alves
Jürgen
J
D020816:Amino Acid Motifs; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004355:Drug Stability; D006868:Hydrolysis; D007700:Kinetics; D008565:Membrane Proteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D014443:Tyrosine / Q000737:chemistry
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050825:Synaptosomal-Associated Protein 25; D014443:Tyrosine; D001120:Arginine; D019274:Botulinum Toxins, Type A