Cleavage of the beta-lactam ring of benzylpenicillin (1) by a zinc(II) complex of the macrocyclic tetraamine 1,4,7,10-tetraazacyclododecane (cyclen) (8) has been studied in aqueous solution as a functional model of a zinc-containing hydrolytic enzyme, beta-lactamase II. beta-Lactam hydrolysis by 8 yielding (5R)-benzylpenicilloate (9) is a second-order reaction (the second-order rate constant k is (4.1 +/- 0.1) x 10(-2) M(-1) s(-1) at 25 degrees C and I = 0.10 (NaNO3)), and a plot of the hydrolysis rate vs pH (6.6-9.6) gives a sigmoidal curve with an inflection point at pH 7.9, which is identical to the pK(a) value for the Zn-II-bound water of Zn-II-cyclen-OH2 (8a). Thus, Zn-II-cyclen-OH- (8b) must play a crucial role in the hydrolysis of the beta-lactam. The activation energy E(a) for the hydrolysis by 8b was determined to be 49 kJ mol(-1), lower than the value of 61 kJ mol(-1) by aqueous OH- ion. The lower E(a) value for 8b is due to the acidic nature of Zn-II that stabilizes the anionic tetrahedral intermediate 15. The hydrolysis is subject to anion inhibition by deprotonated succinimide(-), SCN-, CH3COO-, or Cl- in the same order as their binding affinity for 8a. The Zn-II-cyclen 8 simultaneously catalyzes the isomerization of (5R)-benzylpenicilloate (9) to the 5S-epimer 10 at pH 6.5-9.5. The pH dependency of the catalytic activity discloses that the reactive species is 8a and the kinetically obtained pK(a) value of 8.0 is almost the same as that obtained thermodynamically. The second-order rate constant k(ep) with 8a is 2.5 +/- 0.1 M(-1) s(-1) at 25 degrees C and I = 0.10 (NaNO3). Presumably the reaction involves C-S bond rupture due to coordination of Zn-II-cyclen, followed by recombination to form 10. While the reaction mechanism of beta-lactamase II is still not known in detail, analogies may be drawn with the common role of the Zn-II-OH- species in other zinc enzymes such as carbonic anhydrase.