Correction to Kinetic Study of the Effect of pH on the Trypsin-Catalyzed Hydrolysis of -α-benzyloxycarbonyl-l-lysine--nitroanilide: Mechanism of Trypsin Catalysis.

[This corrects the article DOI: 10.1021/acsomega.9b03750.

Kinetic Studies of the Effect of pH on the Trypsin-Catalyzed Hydrolysis of -α-benzyloxycarbonyl-l-lysine--nitroanilide: Mechanism of Trypsin Catalysis.

The pH dependence of the trypsin-catalyzed hydrolysis of -α-benzyloxycarbonyl-l-lysine -nitroanilide has been studied at 25 °C. / was maximal at alkaline pH values but decreased with decreasing pH. / was dependent on free enzyme p values of 6.

A new lysine derived glyoxal inhibitor of trypsin, its properties and utilization for studying the stabilization of tetrahedral adducts by trypsin.

New trypsin inhibitors Z-Lys-COCHO and Z-Lys-H have been synthesised. values for Z-Lys-COCHO, Z-Lys-COOH, Z-Lys-H and Z-Arg-COOH have been determined. The glyoxal group (-COCHO) of Z-Lys-COCHO increases binding ~300 fold compared to Z-Lys-H.

Structural and biochemical properties of LuxF from Photobacterium leiognathi.

The lux-operon of bioluminescent bacteria contains the genes coding for the enzymes required for light emission. Some species of Photobacteria feature an additional gene, luxF, which shows similarity to luxA and luxB, the genes encoding the heterodimeric luciferase. Isolated dimeric LuxF binds four molecules of an unusually derivatized flavin, i.

Quantifying tetrahedral adduct formation and stabilization in the cysteine and the serine proteases.

Two new papain inhibitors have been synthesized where the terminal α-carboxyl groups of Z-Phe-Ala-COOH and Ac-Phe-Gly-COOH have been replaced by a proton to give Z-Phe-Ala-H and Ac-Phe-Gly-H. We show that for papain, replacing the terminal carboxylate group of a peptide inhibitor with a hydrogen atom decreases binding 3-4 fold while replacing an aldehyde or glyoxal group with a hydrogen atom decreases binding by 300,000-1,000,000 fold. Thiohemiacetal formation by papain with aldehyde or glyoxal inhibitors is shown to be ~10,000 times more effective than hemiacetal or hemiketal formation with chymotrypsin.

A 13C-NMR study of azacryptand complexes.

An azacryptand has been solubilised in aqueous media containing 50% (v/v) dimethyl sulphoxide. (13)C-NMR has been used to determine how the azacryptand is affected by zinc binding at pH 10. Using (13)C-NMR and (13)C-enriched bicarbonate we have been able to observe the formation of 4 different carbamate derivatives of the azacryptand at pH 10.

Hemiacetal stabilization in a chymotrypsin inhibitor complex and the reactivity of the hydroxyl group of the catalytic serine residue of chymotrypsin.

The aldehyde inhibitor Z-Ala-Ala-Phe-CHO has been synthesized and shown by (13)C-NMR to react with the active site serine hydroxyl group of alpha-chymotrypsin to form two diastereomeric hemiacetals. For both hemiacetals oxyanion formation occurs with a pKa value of ~7 showing that chymotrypsin reduces the oxyanion pKa values by ~5.6 pKa units and stabilizes the oxyanions of both diastereoisomers by ~32kJmol(-1).

Vinyl sulfone-based peptidomimetics as anti-trypanosomal agents: design, synthesis, biological and computational evaluation.

A series of vinyl sulfone-containing peptidomimetics were rationally designed, synthesized, and evaluated against Trypanosoma brucei brucei . These electrophilic compounds are likely to exert their antitrypanosomal activity via inhibition of trypanosomal cysteine proteases, TbCatB and rhodesain, through alkylation of a key cysteine residue within the protease active site. The series was designed to present complementary groups to naturally recognized peptide substrates while probing tolerance to a range of substitutions at the P1, P1', and P2 positions.

Importance of tetrahedral intermediate formation in the catalytic mechanism of the serine proteases chymotrypsin and subtilisin.

Two new inhibitors in which the terminal α-carboxyl groups of Z-Ala-Ala-Phe-COOH and Z-Ala-Pro-Phe-COOH have been replaced with a proton to give Z-Ala-Ala-Phe-H and Z-Ala-Pro-Phe-H, respectively, have been synthesized. Using these inhibitors, we estimate that for α-chymotrypsin and subtilisin Carlsberg the terminal carboxylate group decreases the level of inhibitor binding 3-4-fold while a glyoxal group increases the level of binding by 500-2000-fold. We show that at pH 7.

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions.

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.

pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor.

The stromelysin-1 catalytic domain(83-247) (SCD) is stable for at least 16 h at pHs 6.0-8.4.

Conformational, receptor interaction and alanine scan studies of glucose-dependent insulinotropic polypeptide.

Glucose-dependent insulinotropic polypeptide (GIP) is an insulinotropic incretin hormone that stimulates insulin secretion during a meal. GIP has glucose lowering abilities and hence is considered as a potential target molecule for type 2 diabetes therapy. In this article, we present the solution structure of GIP in membrane-mimicking environments by proton NMR spectroscopy and molecular modelling.

Oxyanion and tetrahedral intermediate stabilisation by subtilisin: detection of a new tetrahedral adduct.

The peptide-derived glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal has been shown to be approximately 10 fold more effective as an inhibitor of subtilisin than Z-Ala-Pro-Phe-glyoxal. Signals at 107.2 ppm and 200.

Prolonged L-alanine exposure induces changes in metabolism, Ca(2+) handling and desensitization of insulin secretion in clonal pancreatic beta-cells.

Acute insulin-releasing actions of amino acids have been studied in detail, but comparatively little is known about the beta-cell effects of long-term exposure to amino acids. The present study examined the effects of prolonged exposure of beta-cells to the metabolizable amino acid L-alanine. Basal insulin release or cellular insulin content were not significantly altered by alanine culture, but acute alanine-induced insulin secretion was suppressed by 74% (P<0.

Determination of the structure of tetrahedral transition state analogues bound at the active site of chymotrypsin using 18O and 2H isotope shifts in the 13C NMR spectra of glyoxal inhibitors.

The peptide-derived glyoxal inhibitor Z-Ala-Pro-Phe-glyoxal, where Z is benzyloxycarbonyl, is an extremely potent inhibitor of chymotrypsin. When it is bound to chymotrypsin both the glyoxal (RCOCHO) keto and aldehyde carbons are sp3 hybridized with chemical shifts of 100.7 and 91.

NMR study of the inhibition of pepsin by glyoxal inhibitors: mechanism of tetrahedral intermediate stabilization by the aspartyl proteases.

Z-Ala-Ala-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of pepsin with a Ki = 89 +/- 24 nM at pH 2.0 and 25 degrees C. Both the ketone carbon (R13COCHO) and the aldehyde carbon (RCO13CHO) of the glyoxal group of Z-Ala-Ala-Phe-glyoxal have been 13C-enriched.

13C- and 1H-NMR studies of oxyanion and tetrahedral intermediate stabilization by the serine proteinases: optimizing inhibitor warhead specificity and potency by studying the inhibition of the serine proteinases by peptide-derived chloromethane and glyoxal inhibitors.

Catalysis by the serine proteinases proceeds via a tetrahedral intermediate whose oxyanion is stabilized by hydrogen-bonding in the oxyanion hole. There have been extensive (13)C-NMR studies of oxyanion and tetrahedral intermediate stabilization in trypsin, subtilisin and chymotrypsin using substrate-derived chloromethane inhibitors. One of the limitations of these inhibitors is that they irreversibly alkylate the active-site histidine residue which results in the oxyanion not being in the optimal position in the oxyanion hole.

The bioactive conformation of glucose-dependent insulinotropic polypeptide by NMR and CD spectroscopy.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal incretin hormone, which modulates physiological insulin secretion. Because of its glucose-sensitive insulinotropic activity, there has been a considerable interest in utilizing the hormone as a potential treatment for type 2 diabetes. Structural parameters obtained from NMR spectroscopy combined with molecular modeling techniques play a vital role in the design of new therapeutic drugs.

13C and 1H NMR studies of ionizations and hydrogen bonding in chymotrypsin-glyoxal inhibitor complexes.

Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-Ala-Phe-glyoxal have both been shown to be inhibitors of alpha-chymotrypsin with minimal Ki values of 19 and 344 nM, respectively, at neutral pH. These Ki values increased at low and high pH with pKa values of approximately 4.0 and approximately 10.

Investigation of the effects of sulfonylurea exposure on pancreatic beta cell metabolism.

Prolonged exposure of pancreatic beta cells to the sulfonylureas glibencamide and tolbutamide induces subsequent desensitization to the actions of these drugs. The precise mechanisms underlying this desensitization remain unknown, prompting the present study, which investigated the impact of prolonged sulfonylurea exposure on glucose and energy metabolism using clonal pancreatic BRIN-BD11 beta cells. Following prolonged exposure to tolbutamide, BRIN-BD11 beta cells were incubated in the presence of [U-(13)C]glucose, and isotopomer analysis revealed that there was a change in the ratio of flux through pyruvate carboxylase (EC 6.

Effect of acute dietary standardization on the urinary, plasma, and salivary metabolomic profiles of healthy humans.

Background: Metabolomics in human nutrition research is faced with the challenge that changes in metabolic profiles resulting from diet may be difficult to differentiate from normal physiologic variation.

Objective: We assessed the extent of intra- and interindividual variation in normal human metabolic profiles and investigated the effect of standardizing diet on reducing variation.

Design: Urine, plasma, and saliva were collected from 30 healthy volunteers (23 females, 7 males) on 4 separate mornings.

NMR and alanine scan studies of glucose-dependent insulinotropic polypeptide in water.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that stimulates the secretion of insulin after ingestion of food. GIP also promotes the synthesis of fatty acids in adipose tissue. Therefore, it is not surprising that numerous literature reports have shown that GIP is linked to diabetes and obesity-related diseases.

Impact of the gliotoxin L-serine-O-sulphate on cellular metabolism in cultured rat astrocytes.

L-serine-O-sulphate is a member of a group of amino acids collectively called gliotoxins and is a substrate for the high affinity sodium-dependent glutamate transporters. Previous studies have shown that it is toxic to primary cultures of astrocytes but the mode of toxicity is unknown. The current study demonstrates that L-serine-O-sulphate, at a sub-toxic concentration (400 microM), causes significant disruption to glucose and alanine metabolism in cultures of rat cortical astrocytes.

Ionisations within a subtilisin-glyoxal inhibitor complex.

Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of subtilisin with a K(i)=2.3+/-0.2 microM at pH 7.

NMR structure of the glucose-dependent insulinotropic polypeptide fragment, GIP(1-30)amide.

Glucose-dependent insulinotropic polypeptide is an incretin hormone that stimulates insulin secretion and reduces postprandial glycaemic excursions. The glucose-dependent action of GIP on pancreatic beta-cells has attracted attention towards its exploitation as a potential drug for type 2 diabetes. Use of NMR or X-ray crystallography is vital to determine the three-dimensional structure of the peptide.