In our preliminary studies, my group has collected kinetic data for E. coli RTEM-2 Beta-lactamase. This was done in order to understand the mechanism of action of this enzyme. We studied the substrate specificity of this Class A B-lactamase for the two cephalosporins PADAC and nitrocefin. We found the presence of essential groups on the free enzyme which have pKs of 5.5 and 7.5. These pKs are tentatively assigned to the active groups Glu-166 and Lys-73. pK2 was shifted by substrate binding, while pK1 was unchanged. Inhibition of E. coli RTEM-2 B-lactamase by boronic acids was rapid, occurring within a minute of mixing. Of the tested boronic acids, the arylboronic acids with bulky hydrophobic substituents were the most potent inhibitors. Of the other substituted benzeneboronic acids, those with electron-withdrawing substituents wee better inhibitors. The pH-dependence of inhibition for this group of compounds showed the ionization of a basic group with a pK of about 5.3. pK2 reflected the ionization of the substituted benzeneboronic acid. In this proposal, I propose to give MBRS students the opportunity to prepare a potential pair of transition state analog inhibitors of the B- lactamases, a boronic acid and an analogous aldehyde. Boronic acids have l-already been shown to be good inhibitors; with aldehydes the picture is less clear. A specific aldehyde should clarify if aldehydes can inhibit these serine enzymes as they do serine proteases. Also, A specific boronic acid inhibitor can help clarify or view of the enzyme's mechanism.