The aim of this study was to find a lipase suitable as a surrogate for Human Gastric Lipase (HGL), since the development of predictive gastrointestinal lipolysis models are hampered by the lack of a lipase with similar digestive properties as HGL. Three potential surrogates for HGL; Rhizopus Oryzae Lipase (ROL), Rabbit Gastric Lipase (RGL) and recombinant HGL (rHGL), were used to catalyze the in vitro digestion of two infant formulas (a medium-chain triacylglyceride enriched formula (MC-IF) and a predominantly long-chain triacylglyceride formula (LC-IF)). Digesta were withdrawn after 0, 5, 15, 30, 60 min of gastric digestion and after 90 or 180 min of intestinal digestion with or without the presence of pancreatic enzymes, respectively. The digesta were analyzed by scanning electron microscopy and gas chromatography to quantify the release of fatty acids (FAs). Digestions of both formulas, catalyzed by ROL, showed that the extent of gastric digestion was higher than expected from previously published in vivo data. ROL was furthermore insensitive to FA chain length and all FAs were released at the same pace. RGL and rHGL favoured the release of MC-FAs in both formulas, but rHGL did also release some LC-FAs during digestion of MC-IF, whereas RGL only released MC-FAs. Digestion of a MC-IF by HGL in vivo showed that MC-FAs are preferentially released, but some LC-FAs are also released. Thus of the tested lipase rHGL replicated the digestive properties of HGL the best and is a suitable surrogate for HGL for use in in vitro gastrointestinal lipolysis models.

Synthesis of dTMP by thymidylate synthase proceeds by the reductive methylation of dUMP, which is obtained via one of two parallel pathways. One pathway, considered to be a minor supplier of dTTP [1][2][3], involves the reduction of UDP (UTP) by the action of ribonucleotide reductase. Subsequently, dUDP is phosphorylated to dUTP and cleaved to dUMP. The main supply of dUMP, however, involves the deamination The trimeric dCTP deaminase produces dUTP that is hydrolysed to dUMP by the structurally closely related dUTPase. This pathway provides 70-80% of the total dUMP as a precursor for dTTP. Accordingly, dCTP deaminase is regulated by dTTP, which increases the substrate concentration for half-maximal activity and the cooperativity of dCTP saturation. Likewise, increasing concentrations of dCTP increase the cooperativity of dTTP inhibition. Previous structural studies showed that the complexes of inactive mutant protein, E138A, with dUTP or dCTP bound, and wild-type enzyme with dUTP bound were all highly similar and characterized by having an ordered C-terminal. When comparing with a new structure in which dTTP is bound to the active site of E138A, the region between Val120 and His125 was found to be in a new conformation. This and the previous conformation were mutually exclusive within the trimer. Also, the dCTP complex of the inactive H121A was found to have residues 120-125 in this new conformation, indicating that it renders the enzyme inactive. The C-terminal fold was found to be disordered for both new complexes. We suggest that the cooperative kinetics are imposed by a dTTP-dependent lag of product formation observed in presteady-state kinetics. This lag may be derived from a slow equilibration between an inactive and an active conformation of dCTP deaminase represented by the dTTP complex and the dUTP ⁄ dCTP complex, respectively. The dCTP deaminase then resembles a simple concerted system subjected to effector binding, but without the use of an allosteric site.Abbreviations E138A, mutant dCTP deaminase with a Glu138 to Ala substitution; H121A, mutant dCTP deaminase with a His121 to Ala substitution; V122G, mutant dCTP deaminase with a Val122 to Gly substitution.

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg 115 and Glu 138 , were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wildtype E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg 2؉ , and the mutant enzyme also with the substrate dCTP and Mg 2؉ . The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg 2؉ that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.The main source of dUMP, the precursor for dTTP in the Gram-negative bacteria Escherichia coli and Salmonella typhimurium, is obtained via a pathway where dCTP is deaminated by dCTP deaminase (EC 3.5.4.13) to yield ammonia and dUTP that subsequently is hydrolyzed by dUTPase to generate dUMP and pyrophosphate (1). In contrast, Gram-positive bacteria and eukaryotic organisms synthesize dTTP from dUMP obtained by deamination of dCMP by the zinc-containing enzyme dCMP deaminase (2). Recently, a bifunctional enzyme from the archaeon Methanocaldococcus jannaschii has been identified (3, 4) that possesses both the dCTP deaminase and dUTPase activities in one polypeptide suggesting that at least in some Archaea, dCTP serves as a source for dUMP. The structure of this archaeal enzyme is now known and the subunit shares an overall fold with dUTPases as well as the organization of subunits in a trimer (5). In the present work we demonstrate that dCTP deaminase from E. coli is yet another member of this family of enzymes, even though significant differences are found in the C-terminal stretch that closes the active site upon catalysis.One very interesting feature of the dCTP deaminase is that the deamination reaction proceeds without aid from a metal cofactor. Other nucleobase or nucleoside deaminases such as cytosine deaminase (6, 7), cytidine deaminase (8), adenosine deaminase, (9) and adenine deaminase (10) all require a catalytic m...

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