The deacylation mechanism in hydrolysis of beta-lactam antibiotics by class A beta-lactamase was investigated by the density functional theory. The model compound was composed of a substrate penicillin G, four catalytic residues(Ser70, Lys73, Ser130, and Glu166), and a water molecule. We have found that the deacylation reaction proceeds via four elementary reactions. First, Lys73 is deprotonated by a concerted double proton transfer from Lys73N to Ser1300gamma and from Ser1300gamma to C3-carboxylate in the substrate. Second, the acyl-enzyme tetrahedral intermediate (TI) is formed by an assistance of Glu166 which acts as a general base catalyst. Third, Lys73 is protonated by concerted double proton transfer from C3-carboxylic acid to Ser1300gamma and from Ser1300gamma to Lys73N. Finally, the degraded substrate is detached from the enzyme in concert with a single proton transfer from Lys73Nzeta to Ser700gamma. It is remarkably noted that the deacylation is not proceeded only by Glu166, but Lys73 also participates in the reaction. Moreover, it has been found that the presence of the C3-carboxyl group of the substrate reduces the barrier, height at the TI formation. This result suggests that the deacylation is a substrate-assisted catalytic process.