beta-Lactamases of classes A and C are believed to have evolved from bacterial enzymes involved in biosynthesis of the peptidoglycan, the so-called penicillin-binding proteins. All these enzymes undergo acylation at an active-site serine by beta-lactam antibiotics as a common feature. However, the fate of the acyl-enzyme species is different for beta-lactamases and penicillin-binding proteins; deacylation is rapid for the former, whereas it is slow for the latter. It is believed that the acquisition of the ability to deacylate the acyl-enzyme intermediate led to the evolution of beta-lactamase activity, which is indispensable for the survival of bacteria in the face of challenge by beta-lactam antibiotics. The mechanisms of deacylation of acyl-enzyme intermediates for beta-lactamases are examined as a means to investigate structural factors in evolutionary descendency of classes A and C of beta-lactamases from penicillin-binding proteins. It is known that in class A beta-lactamases the hydrolytic water approaches the acyl-enzyme intermediate from the alpha-face, a process which is promoted by Glu-166 of these enzymes. An approach from the beta-face for class C beta-lactamase has been proposed. The process of activation of the hydrolytic water is not entirely understood at the present for these enzymes. Two compounds, p-nitrophenyl (2R,5R)-5-prolylacetate (2) and p-nitrophenyl (1S,3S)-3-carboxycyclopentylacetate (3), were synthesized as mechanistic probes to explore whether the hydrolytic water molecule actually approaches the acyl-enzyme species from the beta-face and to investigate a notion that the ring amine at the acyl-enzyme intermediate may promote the hydrolytic reaction. Compound 2 acylates the active site serine of the Q908R beta-lactamase (a class C enzyme), and the intermediate undergoes deacylation. On the other hand, compound 3 only acylates the active site, and not having the requisite amine in its structure, the intermediate resists deacylation. Both compounds serve as substrates for the class A TEM-1 beta-lactamase, as they were expected, since the approach of the hydrolytic water molecule is from the alpha-face in this enzyme and is not promoted by the substrate itself. We conclude that substrate-assisted catalysis applies for the class C beta-lactamases. On the basis of the evidence discussed, the knowledge of the crystal structures for the classes A and C of beta-lactamases and the Steptomyces R61 DD-peptidase/transpeptidase (a PBP), it is proposed herein that evolution of classes A and C of beta-lactamases from a primordial penicillin-binding protein should have been independent events; hence, the process does not represent a linear descendency of one beta-lactamase from the other.