Liliane Dupuis

Crystal structure of human gastric lipase and model of lysosomal acid lipase, two lipolytic enzymes of medical interest.

Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active despite the highly acidic stomach environment. We report here the structure of recombinant human gastric lipase at 3.0-Å resolution, the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists of a core domain belonging to the α/β hydrolase-fold family and a “cap” domain, which is analogous to that present in serine carboxypeptidases. It possesses a classical catalytic triad (Ser-153, His-353, Asp-324) and an oxyanion hole (NH groups of Gln-154 and Leu-67). Four N-glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under a segment consisting of 30 residues, which can be defined as a lid and belonging to the cap domain. The displacement of the lid is necessary for the substrates to have access to Ser-153. A phosphonate inhibitor was positioned in the active site that clearly suggests the location of the hydrophobic substrate binding site. The lysosomal acid lipase was modeled by homology, and possible explanations for some previously reported mutations leading to the cholesterol ester storage disease are given based on the present model.

Cloning, sequencing and further characterization of acylpeptide hydrolase from porcine intestinal mucosa

Acylpeptide hydrolase was purified to homogeneity from porcine intestinal mucosa using a seven-step procedure including ammonium sulfate precipitation, gel filtration as well as anion exchange and affinity chromatography. The specific activity of the enzyme reached 105000 nmol/mg protein per min and the purification was as high as 5500-fold. This tetrameric enzyme is composed of four apparently identical subunits, the molecular mass of which was estimated to be 75 kDa, based on the results of amino acid analysis and gel electrophoresis performed under denaturing conditions. It is likely that the NH(2)-terminal residue may be acetylated, while serine was found to be the COOH-terminal residue. The hydrolytic activity of the enzyme toward N-acetyl-L-alanine p-nitroanilide at the optimum pH value was increased twofold in the presence of the chloride anion. The K(m) value calculated from the kinetics of the hydrolysis of acetylalanyl peptides was found to be 0.7+/-0.1 mM, whereas the V(max) values decreased from 200 to 50 nmol/min per microgram of enzyme, depending on the peptidic chain lengths. The V(max) value of the synthetic substrate (250 nmol/min per microgram of enzyme) was 25-500% higher than those of the acetylalanyl peptides, depending on the peptide chain length, although the enzyme affinity was slightly lower (1.8 mM as compared with 0.7 mM). In line with data on other animal species and on various tissues, the enzyme seemed likely to be a serine protease, since it was readily inhibited by diisopropyl fluorophosphate and diethyl pyrocarbonate. A 2377-nucleotide long cDNA coding for the enzyme was isolated from pig small intestine. The deduced amino acid sequence consisted of 731 residues and showed a single different amino acid with that of the porcine liver APH, except the N-terminal amino acid which is still probably lacking.

Oxidation of methionine and 2-hydroxy 4-methylthiobutanoic acid stereoisomers in chicken tissues

Oxidation of DL-2-hydroxy 4-methylthiobutanoic acid (DL-HMB), DL-methionine (DL-MET) and L-methionine (L-MET) in chicken tissue homogenates was compared using 1-14C-labelled tracers. The pattern of oxidation of the substrates was similar at both low (0.7 mM) and high (20 mM) concentrations. The rate of conversion to 2-keto 4-methylthiobutanoic acid (KMB) was highest for DL-MET and lowest for L-MET in kidney, liver and intestinal mucosa. In breast muscle, rates for DL-MET and L-MET were similar at 0.7 mM, but DL-HMB showed the highest rate at 20 mM. Kidney contained the highest specific activity for oxidation of all three substrates. Raising the pH of liver and kidney homogenates from 7.5 to 8.6 increased the oxidation of DL-MET, exclusively. Experiments with inhibitors of D-2-hydroxy acid dehydrogenase (EC 1.1.99.6) and L-2-hydroxy acid oxidase (EC 1.1.3.15) suggested that D- and L-HMB were stereospecifically oxidized by the enzymes. KMB stimulated L-MET oxidation in kidney yet inhibited L-MET oxidation in liver homogenates. The effect of KMB on DL-MET and DL-HMB oxidation also varied between tissues. Amino-oxyacetate inhibited L-MET oxidation completely and DL-MET and DL-HMB oxidation almost completely in both kidney and liver. L-Cycloserine was less potent than amino-oxyacetate and decreased L-MET oxidation more in kidney than in liver. It can be calculated from the results that, at low substrate concentrations, the liver contributes principally to the whole body oxidation of both DL-HMB and DL-MET. At high (greater than physiological) concentrations, DL-HMB would be oxidized principally in skeletal muscle. At all concentrations, L-MET would be converted to KMB mainly in the muscle.

The N-acylpeptide hydrolase from porcine intestine: isolation, subcellular localization and comparative hydrolysis of peptide and isopeptide bonds.

The N-acylpeptide hydrolase from porcine intestinal mucosa was 2000-fold purified by a five-step procedure. The resulting protein (about 300 kDa) is composed of four apparently identical N-acylated polypeptide chains. The enzyme activity was found to be equally distributed along the crypt-villus axis in the intestine and was characterized as a cytosolic protein. Besides the ability of porcine intestinal APH to cleave the first peptide bond in N-protected peptides (Km: 0.8 mM), it is worth stressing that the enzyme was also found to efficiently catalyze the hydrolysis of the isopeptide bond in N-epsilon-Ac-L-Met-L-Lys (Km: 0.7-1.1 mM). It is suggested that N-acylpeptide hydrolase might not only be involved in the catabolism of intracellular N-acylated protein catabolism but also be responsible for the biological utilization of N-acylated food proteins.

In vitro oxidative decarboxylation of L-2-hydroxy-4-methylthiobutanoic acid by L-2-hydroxy acid oxidase A from chicken liver

The first step in the set of reactions responsible for the biological utilization of L-2-hydroxy-4-methylthiobutanoic acid, the methionine hydroxy analogue, in protein synthesis was investigated in vitro using pure L-2-hydroxy acid oxidase A from chicken liver. The reaction yielded no more than 20% of the corresponding alpha-keto acid, the well-known intermediate in methionine metabolism, and as much as 80% of the subsequent decarboxylation product, 3-methylthiopropionate, suggesting that L-2-hydroxy-4-methylthiobutanoic acid cannot be completely converted into methionine in vivo. It was therefore concluded that chicken liver L-2-hydroxy acid oxidase, a peroxisomal enzyme requiring flavin mononucleotide as a coenzyme, also has an oxidative decarboxylation activity in vitro, which was found to be NADH-dependent. The mechanism possibly underlying the successive conversion of the methionine hydroxy analogue into alpha-keto acid and 3-methylthiopropionate by this NADH:flavin oxidoreductase-decarboxylase activity is described.

Purification and some characteristics of chicken liver L-2-hydroxyacid oxidase A

The isozyme A of L‐2‐hydroxyacid oxidase is a peroxisomal flavoenzyme that catalyzes the oxidation of short‐chain aliphatic L‐2‐hydroxyacids in many tissues of higher organisms. A new purification procedure allowed us to obtain a 1400‐fold purified enzyme from chicken liver. The N‐terminal amino acid of the polypeptide chain was found to be blocked as that of spinach glycolate oxidase, contrastingly with that of rat kidney isozyme B. Its amino acid composition was comparable to that of other known L‐2‐hydroxyacid oxidases. Despite different substrate specificity, some immunological identity was observed between chicken liver L‐2‐hydroxyacid isozyme A and rat kidney isozyme B.

Inhibitory effects of anions and active site amino acid sequence of chicken liver L-2-hydroxyacid oxidase A, a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family

Monocarboxylic acids with aliphatic chains were found to be mixed inhibitors of chicken liver L-2-hydroxyacid oxidase A when L-2-hydroxy-4-methylthiobutanoic acid was used as the substrate. The finding that the binding affinity of the enzyme for monocarboxylic acids was directly proportional to the number of carbon atoms in the chain strongly suggests that in addition to the electrostatic interaction due to the carboxyl moiety, hydrophobic forces may also be involved in the binding affinity of monocarboxylic acids to the enzymes active site. Oxalate, a dicarboxylic acid, also resulted in a mixed-type inhibition of chicken liver L-2-hydroxyacid oxidase A, and, surprisingly, its binding affinity to the enzyme was found to be quite high as compared with monocarboxylic acids. This is probably due to the fact that the two carboxyl groups of oxalate give rise to electrostatic interactions with the positively charged side chains of two adjacent residues in the polypeptide chain. The inhibitory effects of other dicarboxylic acids was found to decrease as the number of carbon atoms in the chain increased. Oxamate was found however to be a novel type of potent inhibitor of the enzyme. All in all, these kinetic studies and the amino acid sequence determination in the active site region after limited proteolysis of the polypeptide chain definitely establish that chicken liver NADH/FMN containing L-2-hydroxyacid oxidase A is a member of the FMN-dependent alpha-hydroxyacid oxidizing enzyme family.

Comparative oxidation rates of methionine hydroxy analogue in rat and chicken kidney

1. L-2-HAOX A and L-2-HAOX B were purified from chicken and rat kidney with purification factors of 550 and 45, respectively. 2. It was established that both enzymes were tetrameric (M(r) = 169,000) and consisted of 40 kDa monomers. 3. While chicken kidney L-2-HAOX A is N-terminally blocked like spinach glycolate oxidase and chicken liver L-2-HAOX A, L-2-HOAX B begins with a Pro residue. 4. The kinetic parameters of L-HMB oxidation by L-2-HAOX A and B were determined. 5. The results are particularly interesting as regards L-HMB oxidation by L-2-HAOX B.