Human pancreatic lipase (HPL) loses more than 80% of its activity when incubated with tetrahydrolipstatin in a buffer containing bile salts. During the inactivation process, large changes are observed in intrinsic tryptophan fluorescence and in the near-ultraviolet circular dichroism. The rate of chemical inactivation is highly comparable to that determined from the time dependence of the spectral changes. It is concluded that HPL undergoes a conformational transition upon inhibitor binding, resulting in a change in the microenvironment of tryptophan residues. Bile salts are needed in this system for effective inactivation of the enzyme by tetrahydrolipstatin, and a large increase in the inactivation rate takes place at about the critical micellar concentration (CMC) of bile salts. The inhibited enzyme can be reactivated by reducing the bile salt concentration to below the CMC, and the changes in tryptophan fluorescence induced by acylation with tetrahydrolipstatin are thereby reversed. This suggests that bile salts above their CMC stabilize the acyl-enzyme complex.Pancreatic lipase is a water-soluble enzyme whch hydrolyses insoluble emulsified dietary triglycerides in the intestinal lumen. The requirements for optimal binding and activity of the lipase at this oil/water interface are uncertain. It has been reported that bile salts present in the duodenal contents inhbit lipase activity 11 -31 and that the activity can be restored by colipase [4-61. Other studies suggest that both bile salts and colipase produce a conformational change that favors binding and catalysis [7]. The X-ray structure of human pancreatic lipase (HPL) shows that the active site is covered by a surface loop and that a conformational change is mandatory to make it accessible for substrate [8]. A model of how a similar surface loop present in the fungal (Rhizomucor rniehei) triacylglycerol lipase could reorientate during binding to the water/oil interface, and induce catalytic activity, has recently been proposed based on the crystal structure of a complex of this enzyme with a phosphonate inhibitor 191.HPL as present in the crystalline state and most likely in aqueous solution is in a closed inactive conformation [8]. Trp252 is part of the flap which covers the Ser152-His263-Asp176 catalytic triad, and its side chain is completely buried. This flap must open before substrate or larger active-site inhibitors can possibly react with the essential Ser1.52. Spectroscopic techniques which are sensitive to changes in tryptophan environments were therefore investigated with the goal of verifying and monitoring the conformational change predicted to occur upon inactivation of HPL with tetrahydro- _ _ _ _ lipstatin (Scheme 1). Tetrahydrolipstatin is a potent inhibitorthat binds covalently via an ester link to the active-site serine of HPL [lo]. Indeed, large changes in intrinsic tryptophan fluorescence, in near-ultraviolet circular dichroism and in fluorescence quenching were observed after reaction of HPL with tetrahydrolipstatin. The rates at wh...

A chemical modification approach was used in this study to identify the active site serine residue of human pancreatic lipase. Purified human pancreatic lipase was covalently modified by incubation with ['HI, ["Cjtetrahydrolipstatin (THL). a potent inhibitor of pancreatic lipase. The radiolabeled lipase was digested with thermolysin, and the peptides were separated by HPLC. A single THL-peptidc-adduct was obtained which was identical to that obtained earlier from porcine pancreatic lipase. This pentapeptide with the sequence VIGHS is covalently bound to a THL molecule via the side chain hydroxyl group of the serine unit corresponding to Ser-152 of the lipase. The selective cleavage of the THL-serine bond by mild acid treatment resulted in the formation of the B-lactone Ro 404441 in high yield snd clearly proves that THL is attached via an ester bond and with retention of stereochemistry at all chiral centers to the side chain hydroxyl group of Ser-152 of the lipase. The results obtained for human pancreatic lipase corroborate the inhibition mechanism of THL found on the porcine enzyme, and are in full agreement with the identification of the Ser-lS2...His-263...Asp-176 catalytic triad in the X-ray structure of human pancreatic lipase.Human pancreatic lipase active-site serine; Tetrahydrolipstatin (THL); Structure of lipase-THL adduct

The proteolytic specificity and activity of a recombinant chymosin (Maxiren) and porcine pepsin on buffalo, cow, and goat whole casein (CN) and β-CN were studied by analyzing the degradation products. The results suggest that the hydrolysis of whole casein of buffalo and goat by chymosin was similar to that of cow casein resulting in αs1-I and β-I, -II, and -III as degradation fragments of αs1- and β-CN. The exception was goat β-I which was resistant to further hydrolysis by chymosin but not to porcine pepsin at pH 5.4−6.2. Increasing NaCl concentration to ≥5% reduced the proteolysis of β-CN in all three species, but not that of αs1-CN. The fragments of β-I, -II, and -III produced from β-CN of the three species gave identical results with PAGE. αs1-I and its degradation fragments had in all three species, regardless of the different electrophoretic mobilities on PAGE, the same sequence of appearance. The results indicate that chymosin and porcine pepsin attacked in buffalo and goat caseins the same regions as known for cow αs1- and β-CN. Keywords: Proteolysis; chymosin; porcine pepsin; buffalo, cow, goat caseins

The impact of glucose on glycerol metabolism, especially on 3-hydroxypropionaldehyde (3-HPA) accumulation by resting cells of Lactobacillus reuteri has been investigated. Two systems were used in the study: MRS(-) (modified MRS - omitting glucose, acetate and Tween 80) and distilled water (H(2)O). In MRS(-), addition of glucose enhanced glycerol metabolism in resting cells of L. reuteri, consequently increasing the accumulation of 3-HPA by regulating the NAD/NADH ratio. Enhanced glycerol metabolism correlated positively with the concentration of glucose. NADH produced during glucose metabolism was preferentially reoxidized to NAD by the reduction of 3-HPA to 1,3-propanediol; an adequate supply of glycerol therefore outweighed the repression of glucose on the accumulation of 3-HPA. At a molar ratio of glucose to glycerol no greater than 0.33, accumulation of 3-HPA was favored. In non-growing medium (H(2)O), addition of glucose seemed to be counter-productive with respect to 3-HPA accumulation. Lactate had a positive impact on glycerol metabolism, presumably by altering the redox flux, resulting in enhanced 3-HPA accumulation in both MRS(-) and H(2)O systems.

3-Hydroxypropionaldehyde (3-HPA) is considered as a potent antimicrobial substance. Exploration of its application as a food preservative or as a therapeutic auxiliary agent has been documented in the literature. In the present work, factors that may impact on 3-HPA accumulation by Lactobacillus reuteri and on the stability of 3-HPA were investigated. Three media - H(2)O, milk and MRS broth - were chosen as test systems. Data indicated that 3-HPA accumulation in resting cells of L. reuteri in a two-step fermentation is greatly affected by temperature, pH, cell age and biomass as well as components in the test system. Within 2 h of incubation, 170 mM 3-HPA could be produced with a cell dry weight of 30 g/l, representing 85% of the glycerol supplied (200 mM) in H(2)O. The presence of glycerol during cell growth increased the productivity of 3-HPA by resting cells. In general, 3-HPA is much more stable in H(2)O than in milk and MRS. Factors that enhanced accumulation of 3-HPA did not simply show the same positive impact on the stability of 3-HPA. Thus, for defined applications, factors affecting production and stability of 3-HPA should be evaluated separately.