Histidine 45 in HpaI was replaced with alanine (H45A) and glutamine (H45Q). In the aldol cleavage reaction, kcat values were lowered by 78- and 2059-fold while Km values were increased by 100- and 42-fold in H45A and H45Q, respectively, compared to the wild-type enzyme. Both mutants displayed higher dissociation constants towards the metal cofactor, pyruvate and the transition state analogue, oxalate. Pyruvate proton exchange rates are consequently reduced in H45A and H45Q. pKa for a catalytic base (6.5) is lost in the mutant enzymes and catalysis is dependent on hydroxide ions. The results show that histidine 45 is important for metal cofactor binding and for facilitating C4-OH proton abstraction of the substrate in the reaction mechanism.