Purification procedure for dipeptidyl peptidase III (DPP III) from human erythrocytes cytosol, entailing separations on DEAE-cellulose, hydroxylapatite and Sephacryl S-200 column, which gave homogeneous preparation in 35% yield, is described. The enzyme was shown to be a monomeric acidic protein (Mr approximately 82,000, pI approximately 4.5-4.6), sensitive to freezing and temperatures above 40 degrees C. It was inhibited by metallo-chelators and sulphydryl reagents, the activity being restored by divalent cations and thiol compounds. Co2 and Zn2 at low concentrations activated the enzyme, most probably by binding at the same site. Co2 prevented DPP III inactivation by di(4-pyridyl)disulfide, indicating that it is a metallo-peptidase with essential SH-groups which might be near or at the binding site for the metal. Among various naphthylamides Arg-Arg-2-naphthylamide was the best substrate (Km = 7.7 microM, kcat = 28 s-1) of the enzyme. DPP III from human erythrocytes hydrolysed also tri- to decapeptides of different composition, provided they did not have proline at P1 or P'1 position. A hydrophobic residue at P'1 was preferred. Among substrates were angiotensins and Leu-enkephalin. The enzyme showed particularly high affinity for angiotensin III.

No abstract

Dipeptidyl peptidase (DPP III) was purified from rat and human erythrocytes using an identical procedure. Electrophoretic analyses revealed the same molecular size and pI for both enzymes. The molecular mass of the human enzyme, measured by matrix-assisted laser desorption/ionization MS, was 82500+/-60 Da. Its tryptic peptide mass profile was determined using the same technique, and the amino acid sequence of two internal peptides was obtained by tandem MS and Edman degradation. A search of databases revealed a high similarity between the human erythrocyte and rat liver DPP III: 21 matches out of 34 detected peptides were found, covering 40% of the total sequence. Matched peptides included the peptide harboring the characteristic HELLGH sequence motif, and a stretch of 19 identical amino acids, containing Glu, a putative ligand of active site zinc. Both enzymes preferred Arg-Arg-2-naphthylamide, and were activated by micromolar Co2+, differing in their pH optima and kcat/Km. Zn2+ ions, sulfhydryl reagents, and aminopeptidase inhibitors, especially probestin, inhibited the rat DPP III more potently. The two enzymes showed the highest affinity for angiotensin III (Ki < 1 microM) and a preference for ahydrophobic residue at the P1' site. However, significant differences in the binding constants for several peptides indicated non-identity in the active site topography of human and rat erythrocyte DPP III.

No abstract

In a series of four racemic phenoxyalkyl-alkyl carbinols, 1-phenoxy-2-hydroxybutane (1) is enantioselectively acetylated by Burkholderia cepacia (formerly Pseudomonas cepacia) lipase with an E value $ 200, whereas for the other three racemates E was found to be # 4. To explain the high preference of B. cepacia lipase for (R)-(1)-1, a precursor of its transition state analogue with a tetrahedral P-atom, (R P ,S P )-O-(2R)-(1-phenoxybut-2-yl)-methylphosphonic acid chloride was prepared and crystallized in complex with B. cepacia lipase. The X-ray structure of the complex was determined, allowing to compare the conformation of the inhibitor with results of molecular modelling.Keywords: Burkholderia/Pseudomonas cepacia lipase, racemic sec alcohols, transition state (TS) analogue, crystal structure, molecular modelling.Among more than 30 commercially available lipases [1][2][3][4] that are frequently used in enantioselective acylation of alcohols and amines [5,6] or in esterification of carboxylic acids and hydrolysis of their esters [7,8], Burkholderia cepacia (formerly Pseudomonas cepacia) lipase is one of the most thoroughly studied. The X-ray crystal structures of the native [9,10], and inhibited [11,12] enzyme in open conformations were reported. These structural data have been used in computer simulations of enantioselective ester hydrolysis catalysed by B. cepacia lipase [13][14][15]. Other relevant reports comprise the study of electronic effects of substituents on the enantioselectivity of B. cepacia lipase [16] and flexible docking based on structural information of inhibited B. cepacia lipase [17].Models for predicting enantiopreference in lipasecatalysed acylations of sec alcohols have been proposed based on experimental results. Kazlauskas et al. [18] proposed an empirical rule that predicts which enantiomer reacts faster. This rule relates the relative size of the substituents on the stereogenic centre with enantioselectivity. Other authors reported results related to this rule, in particular the effect of large- [19] and medium-sized substituents [20]. However, these rules do not clarify the detailed mechanism of the enantiorecognition.For some time we have been studying lipase catalysed stereoselective acylations [21-24] of conformationally flexible sec alcohols as substrates. Two important results emerged. First, not all lipases acylate macrocyclic, sterically more constrained sec alcohols with higher stereoselectivity compared to their open-chain counterparts [24]. Second, some lipases acylate with increased enantioselectivity acyclic sec alcohols with perturbing L (large) and M (medium) groups at larger distance from the stereogenic centre [23]. In this latter case, a nonmonotonous correlation between E value and the distance (n) of the perturbing groups in 1-4 was observed (Fig. 1).Continuing this project, we have undertaken biocatalytic, structural, and modelling studies to get more defined information on the mechanism that provides a high degree or bias of enantioselectivity in acetylation of ...