Carbonic anhydrase activators: Kinetic and X-ray crystallographic study for the interaction of d- and l-tryptophan with the mammalian isoforms I–XIV Original Research Article

An activation study of mammalian carbonic anhydrase (CA, EC 4.2.1.1) isoforms I–XIV with d- and l-tryptophan has been performed both by means of kinetic and X-ray crystallographic techniques. These compounds show a time dependent activity against isozyme CA II, with activation constants of 1.13 μM for l-Trp and 0.37 μM for d-Trp, respectively, after 24 h of incubation between enzyme and activator. The high resolution X-ray crystal structure of the hCA II—d-Trp adduct revealed the activator to bind in a totally unprecedented way to the enzyme active site as compared to histamine, l-/d-Phe, l-/d-His or l-adrenaline. d-Trp is anchored at the edge of the CA II active site entrance, strongly interacting with amino acid residues Asp130, Phe131 and Gly132 as well as with a loop of a second symmetry related protein molecule from the asymmetric unit, by means of hydrogen bonds and several weak van der Waals interactions involving Glu234, Gly235, Glu236 and Glu238. Thus, a second activator binding site (B) within the CA II cavity has been detected, where only d-Trp was shown so far to bind, in addition to the activator binding site A, in which histamine, l-/d-Phe, and l-/d-His are bound. These findings explain the strong affinity of d-Trp for CA II and may be useful for designing novel classes of CA activators by using this compound as lead molecule.