Arg362 and Tyr365 of the Botulinum Neurotoxin Type A Light Chain Are Involved in Transition State Stabilization

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Abstract
The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn2+ is coordinated by His222 and His226 of the Zn2+ binding motif HisGluXXHis and Glu261, whereas Glu223 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 Arg362 and Tyr365 of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu350 which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg362 and substitution of Tyr365 with Phe resulted in 79- and 34-fold lower kcat/Km values, respectively. These changes were provoked by decreased catalytic rates (kcat) and not by alterations of ground state substrate binding as evidenced by largely unchanged Kd and Km values. None of these mutations affected the overall secondary structure or zinc content of the LC. These findings suggest that the guanidino group of Arg362 and the hydroxyl group of Tyr365 together accomplish transition state stabilization as was proposed for thermolysin, being the prototypical member of the gluzincin superfamily of metalloproteases. Mutation of Glu350 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 Zn2+ binding affinity. Glu350 apparently occupies a central position in the active site and presumably positions His222 and Arg362.