FACTORS GOVERNING INTRINSIC CHEMICAL REACTIVITY DIFFERENCES BETWEEN CLAVULANIC AND PENICILLANIC ACID AND ZINC PDB SURVEY: ANALYSES OF ZINC BINDING SITES IN PROTEIN CRYSTAL STRUCTURES

• Main Authors: Yen-lin Lin, 林妍伶
• Other Authors: Carmay Lim
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/98939094764005850254

碩士 === 國立清華大學 === 化學系 === 90 === To help elucidate why penicillin-G is inhibited by certain bacterial -lactamase enzymes, whereas clavulanic acid (Clav, which is similar to penicillin-G except at positions 1, 2 and 6) relieves this inhibition, the intrinsic chemical reactivity of these two antibiotics were assessed in this work. Ab initio and continuum dielectric methods were used to map out the gas-phase and solution-phase free energy profiles for the alkaline hydrolyses of Clav and penicillanic acid (Peni, which is similar to penicillin-G except at position 6) as well as a fictitious hybrid compound, Peni-db, which is similar to Clav and Peni except at position 1 and 2, respectively. Furthermore, ring strain energies of various lactam rings and the five-membered ring of Peni and Clav as well as their respective rate-limiting transition states were computed to assess the contribution of four- and five-membered ring strain to the antibiotic’s activity. The predicted product distribution, rate-limiting step, and relative reaction rates for the alkaline hydrolysis of Peni and Clav are in accord with experiment. The rate-limiting step in the alkaline hydrolysis of Peni, Clav or Peni-db is the approach of the negatively charged hydroxide ion toward the anionic reactant to form a tetrahedral intermediate. Alkaline hydrolysis of Clav generates more stable products than that of Peni because the hydroxyethylidene group in Clav facilitates rotation about the C2C3 bond to yield an intermediate where the amide proton is close to the O1 atom, which can abstract it easier than the less polar S1. Clav undergoes basic hydrolysis faster than Peni mainly because its hydroxyethylidene group increases the positive charge on the carbonyl C7 atom in the rate-limiting transition state (but not in the ground state), therefore enhancing favorable electrostatic interactions with the incoming hydroxide anion. To a lesser extent, the oxygen at position 1 in Clav also contributes to the rate acceleration due to greater solvent stabilization of the oxygen-containing transition state as compared to the respective ground state. Inherent strain of the four-membered -lactam ring or five-membered ring does not enhance the alkaline hydrolyses of -lactam molecules such as Peni or Clav, consistent with the observation that the rate-limiting step does not involve breakdown of the four-membered -lactam ring or five-membered thiazolidine/oxazolidine ring. The geometrical properties of zinc-binding sites in high quality protein X-ray structures deposited in the Protein Data Bank have been examined to identify differences between zinc sites that are directly involved in catalysis (catalytic zinc sites) and those that play only a structural role (structural zinc sites). Ligands in appearing in both structural and catalytic zinc ions are also statistically including. To this end, trends in the 1st- and 2nd-coordination shell were obtained separately for structural and catalytic zinc ions in PDB structures. As expected, the Zn2+ PDB survey shows significant differences between structural and catalytic zinc sites. For structural zinc ion the most common 1st-shell ligand is Cys, whereas for catalytic zinc ions the most abundant zinc-bound ligand is His. The partners for 1st-shell His also differ according to the role/function of zinc. For structural zinc sites, the most abundant His partner is the backbone carbonyl oxygen, whereas for catalytic zinc sites it is the Asp/Glu carboxylate sidechain. For the 1st-shell Cys in structural zinc sites, its most frequent partner in the outer layer is the backbone peptide group. Altogether, for structural zinc sites, the backbone peptide groups dominate the 2nd-shell coordination layer, and [Cys/His]:[BKB] hydrogen bonds could stabilize zinc cores. For catalytic zinc sites, [His]:[Asp/Glu] hydrogen bonds are ubiquitous, indicating a possible role of His in catalysis.