Mireille Rivière

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.

Improved purification and biochemical characterization of phospholipase D from cabbage

Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was purified from cabbage leaves. The two step purification procedure involved hydrophobic chromatography on Octyl-Sepharose followed by a Mono-Q/FPLC-column with a total yield of 23% and a purification factor of 1000. A zymographic assay was used to detection of PL D activities at various stages of purification under non denaturing PAGE. The molecular mass was determined to be 90 kDa using the SDS/PAGE method, and 90,200 Da as calculated from the amino acid analysis. The isoelectric point of the enzyme is acidic (pI = 4.7). The amino-acid composition and 29 residues of the NH2-terminal amino-acid sequence were determined.

Enzymes as biosensors. 2. Hysteretic response of chloroplastic fructose-1, 6-bisphosphatase to fructose 2, 6-bisphosphate

Oxidized chloroplastic fructose-bisphosphatase is almost totally inactive at pH 7.5, that is under pH conditions that prevail in the chloroplast stroma. When preincubated for different time periods with fructose 2,6-bisphosphate and assayed in the absence of this ligand, it displays an activity which is directly related to the duration of the preincubation phase. This implies that fructose 2,6-bisphosphate induces enzyme conformers that appear in sequence and may be competent for catalytic activity. Upon desorption of fructose 2,6-bisphosphate the enzyme may retain its active conformation for a time period whose duration depends on magnesium concentration. It thus appears that reduction of the enzyme is not an obligatory prerequisite for its activity. Fructose 2,6-bisphosphate behaves as a competitive inhibitor of the reduced, active enzyme, with respect to the real substrate. When assayed with the oxidized enzyme, however, it behaves as an activator. Moreover the apparent steady-state rate that may be measured experimentally depends on both fructose 2,6-bisphosphate concentration and the direction of a concentration change. The reaction velocity experimentally measured is thus a meta-steady-state rate and depends on the initial conditions of the system. The fructose-bisphosphatase system thus displays, with respect to fructose 2,6-bisphosphate, a hysteresis loop and may then sense whether the concentration of that ligand is increased or decreased. A model has been proposed which allows one to explain these results. This model is based on the view that the substrate and fructose 2,6-bisphosphate compete for the same site of the enzyme and that this latter ligand stabilizes a conformation competent for enzyme activity. After the ligand has been chased away, the enzyme retains the active conformation for a while and slowly relapses to the initial inactive conformation. The time-scale of this slow relaxation overlaps that of the steady state of product appearance and this generates meta-steady-state kinetics, which is dependent on the initial state and therefore on the history of the system.

Crystallization and preliminary crystallographic study of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp)

The plant phospholipase D (PLD) is considered to be a key enzyme involved in various physiological processes such as signal transduction and membrane metabolism. Crystals of the PLD protein from Vigna unguiculata have been produced from the recombinant 768 amino-acid protein. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 157.7, b = 65.6, c = 90.2 A, beta = 111.5 degrees. There is one molecule in the asymmetric unit. Frozen crystals diffract to at least 1.94 A resolution using synchrotron radiation. A search for heavy-atom derivatives using ytterbium and tungstate is currently under way in order to solve the three-dimensional structure.

In vitro lipolysis by human pancreatic lipase is specifically abolished by its inactive forms

In human adults, the enzymatic hydrolysis of dietary fat along the digestive tract is sequentially catalyzed by two main enzymes, human gastric lipase (HGL) and human pancreatic lipase (HPL). Both a chemically inhibited form of HPL as well as an inactive HPL mutant with a glycine residue substituted for its catalytic serine were found to be strong inactivators of HPL activity. In the presence of bile salts, this inhibition was clearly due to competition for colipase. We established that the chemically inhibited HPL, probably in its open conformation, had a much greater affinity for colipase than the closed native form of HPL. These inhibitory effects are quite substantial, because a 0.2-M excess of the chemically inhibited HPL form relative to HPL reduced the catalytic lipolytic activity by 50% in the presence of an equimolar amount of colipase.

Preduodenal Lipases and their Role in Lipid Digestion

Under normal physiological conditions the digestion and absorption of dietary lipids are highly efficiently processed. In humans, the diet generally contains 90 to 120 g of lipids (mostly triacylglycerols), more than 95% of which are absorbed, due to the interplay between the stomach, the small intestine, the liver, and the pancreas (Carey et al., 1983). Several steps can be distinguished in the processing of dietary lipids, including their emulsification, hydrolysis and solubilization, and, last, their uptake into the enterocyte. The emulsification of lipids starts in the stomach and is mediated by physical forces and is facilitated by the partial lipolysis of the dietary lipids (Carey et al., 1983). For a long time, the hydrolysis of dietary triglycerides was thought to begin in the intestinal lumen and to be catalyzed entirely by pancreatic lipase. The stomach was thought to be a transient storage organ, the role of which was limited to mixing lipids with the other nutriments and dispersing them as required. Although many authors observed the occurrence of lipolysis at the preduodenal level in humans and in several other species, the gastric phase of lipolysis was assumed to be negligible and to be of little or no significance in comparison with the intestinal step. Gastric lipolysis was even attributed to pancreatic contamination resulting from a duodeno-gastric reflux. At the beginning of the twentieth century, however, it was observed that gastric juice could hydrolyze fat. In 1901, Volhard stated that gastric lipase was the “ferment” present in gastric juice that was responsible for fat hydrolysis. Finally, the gastric origin of the lipase present in dog gastric juice was established by Hull and Keaton (1917) in dogs with Pavlov stomach under conditions precluding the possibility of any pancreatic contamination.

Thermodynamics and Kinetics of the Interactions of Thioredoxin Fb with Fructose Bisphosphatase from Spinach Chloroplast

One of the most important process involved in the photoregulation of chloroplast enzymes is their oxidoreductive modification by protein mediators known as thioredoxins (1). Different molecular species of thioredoxins from Spinach chloroplast have been purified and characterized (2). The reductive activation of fructose bisphosphatase is due to the reduction of two disulfide bridges followed by a conformation change of the protein (3). Very little was known, till recently about molecular interaction occuring between the enzyme and its protein effector. We discuss here the binding properties and the kinetics of inactivation of fructose bisphosphatase (FBPase) by thioredoxin fb.

Chloroplastic Thioredoxins from Spinach

It is known that several chloroplastic enzymes are activated by light in vivo. This activation is due to the reduction of these enzymes by a protein factor, the thioredoxin. Recently it was shown that there are several forms of thioredoxins (f and m) in the chloroplast (Schurman et al., 1981 and Soulie et al., 1981). We have developed a method for the purification of four thioredoxins from spinach chloroplasts. We describe some molecular characteristics of these proteins and the activation specificity for three chloroplastic enzymes: the fructose 1,6 bisphosphatase (FBPase), the sedoheptulose 1,7 bisphosphatase (SBPase) and the NADP-malate dehydrogenase (NADP-MDH).