CRYSTALLOGRAPHIC STRUCTURE OF A PHOSPHONATE DERIVATIVE OF THE ENTEROBACTER-CLOACAE P99 CEPHALOSPORINASE - MECHANISTIC INTERPRETATION OF A BETA-LACTAMASE TRANSITION-STATE ANALOG

The crystal structure of a complex formed on reaction of the Enterobac ter cloacae P99 cephalosporinase (beta-lactamase) with a phosphonate m onoester inhibitor, m-carboxyphenyl [N-[(p-iodophenyl)acetyl]amino]met hyl]phosphonate, has been obtained at 2.3-Angstrom resolution. The str ucture shows that the inhibitor has phosphonylated the active site ser ine (Ser64) with loss of the m-carboxyphenol leaving group. The inhibi tor is positioned in the active site in a way that can be interpreted in terms of a transition-state analog. The arylacetamido side chain is placed as anticipated from analogues beta-lactamoyl complexes of peni cillin-recognizing enzymes, with the amido group hydrogen-bonded to th e backbone carbonyl of Ser318 (of the B3 beta-strand) and to the amide s of Gln120 and Asn152. There is support in the asymmetry of the hydro gen bonding of this side chain to the protein and in the 2-fold disord er of the benzyl group for the considerable breadth in substrate speci ficity exhibited by class C beta-lactamases. One phosphonyl oxygen ato m is in the oxyanion hole, hydrogen-bonded to main-chain NH groups of Ser318 and Ser64, while the other oxygen is solvated, not within hydro gen-bonding distance of any amino acid side chain. The closest active site functional group to the solvated oxygen atom is the Tyr150 hydrox yl group (3.4 Angstrom); Lys67 and Lys315 are quite distant (4.3 and 5 .7 Angstrom, respectively). Rather Tyr150 and Lys67 are more closely a ssociated with Ser640 gamma (2.9 and 3.3 Angstrom). This arrangement i s interpreted in terms of the transition state for breakdown of the te trahedral intermediate in the deacylation step of catalysis, where the Tyr150 phenol seems the most likely general acid. Thus, Tyr150, as th e phenoxide anion, would be the general base catalyst in acylation, as proposed by Oefner et al. [Nature (1990) 343, 284-288]. The structure is compared with that of a similar phosphonate derivative of a class A beta-lactamase [Chen et al. (1993) J. Mol. Biol. 234, 165-178], and mechanistic comparisons are made. The sensitivity of serine beta-lacta mases, as opposed to serine proteinases, toward inhibition by phosphon ate monoanions is supported by electrostatic calculations showing a ne t positive potential only in the catalytic sites of the beta-lactamase s.