The crystal structure of Cam, the prototypic γ-class carbonic anhydrase, reveals active site residues Gln75, Asn73, and Asn 202 previously hypothesized to participate in catalysis. These potential roles were investigated for the first time by kinetic analyses of site-specific replacement variants of the zinc and cobalt forms of Cam. Gln75 replacement variants showed large decreases in kcat/Km relative to wild-type. Further, the Gln75 variants showed a loss of the pKa in pH versus kcat/Km profiles previously attributed to ionization of the metal-bound water yielding the hydroxyl group attacking CO2. These results support the previously proposed role for Gln75 in hydrogen bonding with the catalytic hydroxyl orienting it for attack on CO2. Kinetic analyses of Asn73 variants were consistent with a role in hydrogen bonding with Gln75 to position it for optimal interaction with the catalytic hydroxyl. Kinetic analyses of Asn202 variants showed substantial decreases in k cat/Km relative to the wild-type enzyme supporting the previously hypothesized role in polarizing CO2 and facilitating attack from the metal-bound hydroxyl. On the basis of results presented here, and previously reported structural analyses, we present a catalytic mechanism involving Gln75, Asn73, and Asn202 that also suggests a role for Glu62 not previously recognized. Finally, the results suggest that the γ-, β-, and α-class carbonic anhydrases each independently evolved variations of a fundamental hydrogen bond network essential for catalysis.