Structure of secreted aspartic proteinases from Candida. Implications for the design of antifungal agents.

Pathogens of the genus Candida can cause life threatening infections in immuno-compromised patients. The three-dimensional structures of two closely related secreted aspartic proteinases from C. albicans complexed with a potent (Ki = 0.17 nM) inhibitor, and an analogous enzyme from C. tropicalis reveal variations on the classical aspartic proteinase theme that dramatically alter the specificity of this class of enzymes. The novel fungal proteases present: i) an 8 residue insertion near the first disulfide (Cys45-Cys50, pepsin numbering) that results in a broad flap extending towards the active site; ii) a seven residue deletion replacing helix hN2 (Ser110-Tyr114), which enlarges the S3 pocket; iii) a short polar connection between the two rigid body domains that alters their relative orientation and provides certain specificity; and i.v.) an ordered 12 residue addition at the carboxy terminus. The same inhibitor (A-70450) binds in an extended conformation in the two variants of C. albicans protease, and presents a branched structure at the P3 position. However, the conformation of the terminal methylpiperazine ring is different in the two crystals structures. The implications of these findings for the design of potent antifungal agents are discussed.

Quantum chemical studies of a model for peptide bond formation. 3. Role of magnesium cation in formation of amide and water from ammonia and glycine.

The SN2 reaction between glycine and ammonia molecules with magnesium cation Mg2+ as a catalyst has been studied as a model reaction for Mg(2+)-catalyzed peptide bond formation using the ab initio Hartree-Fock molecular orbital method. As in previous studies of the uncatalyzed and amine-catalyzed reactions between glycine and ammonia, two reaction mechanisms have been examined, i.e., a two-step and a concerted reaction. The stationary points of each reaction including intermediate and transition states have been identified and free energies calculated for all geometry-optimized reaction species to determine the thermodynamics and kinetics of each reaction. Substantial decreases in free energies of activation were found for both reaction mechanisms in the Mg(2+)-catalyzed amide bond formation compared with those in the uncatalyzed and amine-catalyzed amide bond formation. The catalytic effect of the Mg2+ cation is to stabilize both the transition states and intermediate, and it is attributed to the neutralization of the developing negative charge on the electrophile and formation of a conformationally flexible nonplanar five-membered chelate ring structure.

Liquid-chromatographic measurement of creatinine in serum and urine.

We describe the adaptation of a "high-performance" liquid chromatographic method for determination of creatinine in serum and urine. The proposed method is simple, rapid, precise, and accurate. The retention time for creatinine can be varied simply by changing the KH2PO4 concentration in the mobile phase: acetonitrile/aqueous KH2PO4 (1/4 by vol). Within-day precision (CV) was 1.2-3.6% in serum chromatographed with an internal standard, and 2.3-2.8% in serum when an external standard was used. Between-day precision (CV) was 1.3-2.1% in serum and 1.3-2.7% in urine (with an external standard). Analytical recoveries of creatinine added to serum were 94-100% for the method with an internal standard, 95-103% with an external standard.