The susceptibility to tryptic hydrolysis of peptide bonds involving epsilon-N-methyllysine

Using isolation of soluble peptides as a means of comparing the efficiency of tryptic hydrolysis of various forms of the phase-1 flagellin of Salmonella typhimurium, it was found that peptides terminating in N-methyllysine required a longer period of exposure to enzyme for their detection than did equivalent peptides terminating in lysine.     

Thermodynamics of hydrolysis of oligosaccharides

Microcalorimetry has been used to determine enthalpy changes for the hydrolysis of a series of oligosaccharides. High-pressure liquid chromatography was used to determine the extents of reaction and to check for any possible side reactions. The enzyme glucan 1,4-alpha-glucosidase was used to bring about the following hydrolysis reactions: (A) maltose(aq) + H2O(liq) = 2D-glucose(aq); (B) maltotriose(aq) + 2H2O(liq) = 3D-glucose(aq); (C) maltotetraose(aq) + 3H2O(liq) = 4D-glucose(aq); (D) maltopentaose(aq) + 4H2O(liq) = 5D-glucose(aq); (E) maltohexaose(aq) + 5H2O(liq) = 6D-glucose(aq); (F) maltoheptaose(aq) + 6H2O(liq) = 7D-glucose(aq); (G) amylose(aq) + nH2O(liq) = (n + 1) D-glucose(aq); and (H) panose(aq) + 2H2O(liq) = 3D-glucose(aq); (J) isomaltotriose(aq) + 2H2O(liq) = 3D-glucose(aq). The enzyme beta-fructofuranosidase was used for the reactions: (K) raffinose(aq) + H2O(liq) = alpha-D-melibiose(aq) + D-fructose(aq); and (L) stachyose(aq) + H2O(liq) = o-alpha-D-galactopyranosyl-(1----6)- alpha-o-D-galactopyranosyl-(1----6)-alpha-D-glucopyranose + D-fructose(aq). The results of the calorimetric measurements (298.15 K, 0.1 M sodium acetate buffer, pH 4.44-6.00) are: delta H0A = -4.55 +/- 0.10, delta H0B = -9.03 +/- 0.10, delta H0C = -13.79 +/- 0.15, delta H0D = -18.12 +/- 0.10, delta H0E = -22.40 +/- 0.15, delta H0F = -26.81 +/- 0.20, delta H0H = 1.46 +/- 0.40, delta H0J = 11.4 +/- 2.0, delta H0K = -15.25 +/- 0.20, and delta H0L = -14.93 +/- 0.20 kJ mol-1. The enthalpies of hydrolysis of two different samples of amylose were 1062 +/- 20 and 2719 +/- 100 kJ mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of the hydrolysis of sucrose

A thermodynamic investigation of the hydrolysis of sucrose to fructose and glucose has been performed using microcalorimetry and high-pressure liquid chromatography. The calorimetric measurements were carried out over the temperature range 298-316 K and in sodium acetate buffer (0.1 M, pH 5.65). Enthalpy and heat capacity changes were obtained for the hydrolysis of aqueous sucrose (process A): sucrose(aq) + H2O(liq) = glucose(aq) + fructose (aq). The determination of the equilibrium constant required the use of a thermochemical cycle calculation involving the following processes: (B) glucose 1-phosphate2-(aq) = glucose 6-phosphate2-(aq); (C) sucrose(aq) + HPO4(2-)(aq) = glucose 1-phosphate2-(aq) + fructose(aq); and (D) glucose 6-phosphate2-(aq) + H2O(liq) = glucose(aq) + HPO4(2-)(aq). The equilibrium constants determined at 298.15 K for processes B and C are 17.1 +/- 1.0 and 32.4 +/- 3.0, respectively. Equilibrium data for process D was obtained from the literature, and in conjunction with the data for processes B and C, used to calculate a value of the equilibrium constant for the hydrolysis of aqueous sucrose. Thus, for process A, delta G0 = -26.53 +/- 0.30 kJ mol-1, K0 = (4.44 +/- 0.54) x 10(4), delta H0 = -14.93 +/- 0.16 kJ mol-1, delta So = 38.9 +/- 1.2 J mol-1 K-1, and delta CoP = 57 +/- 14 J mol-1 K-1 at 298.15 K. Additional thermochemical cycles that bear upon the accuracy of these results are examined.     

Thermodynamics of hydrolysis of sugar phosphates

Thermodynamics of the enzyme-catalyzed (alkaline phosphatase, EC 3.1.3.1) hydrolysis of glucose 6-phosphate, mannose 6-phosphate, fructose 6-phosphate, ribose 5-phosphate, and ribulose 5-phosphate have been investigated using microcalorimetry and, for the hydrolysis of fructose 6-phosphate, chemical equilibrium measurements. Results of these measurements for the processes sugar phosphate2- (aqueous) + H2O (liquid) = sugar (aqueous) + HPO2++-(4) (aqueous) at 25 degrees C follow: delta Ho = 0.91 +/- 0.35 kJ.mol-1 and delta Cop = -48 +/- 18 J.mol-1.K-1 for glucose 6-phosphate; delta Ho = 1.40 +/- 0.31 kJ.mol-1 and delta Cop = -46 +/- 11 J.mol-1.dK-1 for mannose 6-phosphate; delta Go = -13.70 +/- 0.28 kJ.mol-1, delta Ho = -7.61 +/- 0.68 kJ.mol-1, and delta Cop = -28 +/- 42 J.mol-1.K-1 for fructose 6-phosphate; delta Ho = -5.69 +/- 0.52 kJ.mol-1 and delta Cop = -63 +/- 37 J.mol-1.K-1 for ribose 5-phosphate; and delta Ho = -12.43 +/- 0.45 kJ.mol-1 and delta Cop = -84 +/- 30 J.mol-1.K-1 for the hydrolysis of ribulose 5-phosphate. The standard state is the hypothetical ideal solution of unit molality. Estimates are made for the equilibrium constants for the hydrolysis of ribose and ribulose 5-phosphates. The effects of pH, magnesium ion concentration, and ionic strength on the thermodynamics of these reactions are considered.     

The resistance to tryptic hydrolysis of peptide bonds adjacent to N epsilon,N-dimethyllysyl residues

A peptide from sperm whale myoglobin, residues 132-153, and a chromogenic substrate, H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide diacetate, were selected to investigate the susceptibility of peptide bonds adjacent to N epsilon,N-dimethyllysyl residues to tryptic hydrolysis. The peptides were exhaustively methylated using formaldehyde and sodium cyanoborohydride (N. Jentoft and D. G. Dearborn (1979) J. Biol. Chem. 254, 4359-4365). Unmodified and methylated peptides were digested with trypsin or submaxillary protease, an enzyme that catalyzes the hydrolysis of only arginyl bonds in proteins. Trypsin catalyzed the hydrolysis of the methylated apomyoglobin peptide only at the single arginyl residue and not at any of the four N epsilon,N-dimethyllysyl residues. Trypsin also failed to catalyze the hydrolysis of reductively methylated H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide. Even a 17-fold molar excess of the methylated substrate did not appear to alter the rate of tryptic hydrolysis of the unmodified substrate. These results are discussed with regard to the interactions of substrates within the specificity site of trypsin.     

Evidence for mutual exclusion in the tryptic hydrolysis of two peptide bonds in maleylated trypsinogen

Treatment of completely maleylated trypsinogen with trypsin resulted in a mutual exclusion of hydrolysis of the two arginyl peptide bonds wherein one half of the molecules of the modified zymogen were cleaved at the Arg55-Leu56 bond and the other half of the molecules were cleaved at the Arg105- Val106 bond and no molecules were cleaved at both arginyl bonds.     

How ribosomes make peptide bonds

Ribosomes are molecular machines that synthesize proteins in the cell. Recent biochemical analyses and high-resolution crystal structures of the bacterial ribosome have shown that the active site for the formation of peptide bonds--the peptidyl-transferase center--is composed solely of rRNA. Thus, the ribosome is the largest known RNA catalyst and the only natural ribozyme that has a synthetic activity. The ribosome employs entropic catalysis to accelerate peptide-bond formation by positioning substrates, reorganizing water in the active site and providing an electrostatic network that stabilizes reaction intermediates. Proton transfer during the reaction seems to be promoted by a concerted shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate.     

Partial vapor-phase hydrolysis of peptide bonds: A method for mass spectrometric determination of O-glycosylated sites in glycopeptides

In this study we present a method for determination of O-glycosylation sites in glycopeptides, based on partial vapor-phase acid hydrolysis in combination with mass spectrometric analysis. Pentafluoropropionic acid and hydrochloric acid were used for the hydrolysis of glycosylated peptides. The reaction conditions were optimized for efficient polypeptide backbone cleavages with minimal cleavage of glycosidic bonds. The glycosylated residues were identified by mass spectrometric analysis of the hydrolytic cleavage products. Although glycosidic bonds are partially cleaved under acid hydrolysis, the resulting mass spectra allowed unambiguous determination of the glycosylation sites. Examples are shown with mannosyl- and mucin-type glycopeptides. Performing the hydrolysis in vapor eliminates the risk for contamination of the sample with impurities from the reagents, thus allowing analysis of the reaction products without further purification both by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry.     

Base hydrolysis of phosphodiester bonds in pneumococcal polysaccharides

A comprehensive study of the base hydrolysis of all phosphodiester bond-containing capsular polysaccharides of the 23-valent pneumococcal vaccine is described here. Capsular polysaccharides from serotypes 6B, 10A, 17F, 19A, 19F, and 20 contain a phosphodiester bond that connects the repeating units in these polysaccharides (also referred to as backbone phosphodiester bonds), and polysaccharides from serotypes 11A, 15B, 18C, and 23F contain a phosphodiester bond that links a side chain to their repeating units. Molecular weight measurements of the polysaccharides, using high performance size exclusion chromatography with tandem multiangle laser light scattering and refractive index detection, was used to evaluate the kinetics of hydrolysis. The measurement of molecular weight provides a high degree of sensitivity in the case of small extents of reaction, thus allowing reliable measurements of the kinetics over short times. Pseudo-first-order rate constants for these polysaccharides were estimated using a simple model that accounts for the polydispersity of the starting sample. It was found that the relative order of backbone phosphodiester bond instability due to base hydrolysis was 19A > 10A > 19F > 6B > 17F, 20. Degradation of side-chain phosphodiester bonds was not observed, although the high degree of sensitivity in measurements is lost in this case, due to the low contribution of the side chains to the total polysaccharide molecular weight. In comparison with literature data on pneumococcal polysaccharide 6A, 19A was found to be the more labile, and hence appears to be the most labile pneumococcal polysaccharide studied to date. The rate of hydrolysis increased at higher pH and in the presence of divalent cation, but the extent was lower than expected based on similar data on RNA. Finally, the differences in the phosphodiester bond stabilities were analyzed by considering stereochemical factors in these polysaccharides. These results also provide a framework for evaluation of molecular integrity of phosphodiester-bond-containing polysaccharides in different solution conditions.     

Structural analysis of the peptides derived from specific acid-catalyzed hydrolysis at aspartylprolyl peptide bonds in human J chain.

Human J chain from IgM has been selectively cleaved at three aspartylprolyl peptide bonds to yield four fragments containing 62, 20, 25, and 22 amino acids, respectively. The amino acid sequence of each peptide has been partially determined, (59 of a total of 129 residues) and its position in the J chain ascertained. There were no obvious similarities to known sequences in other immunoglobulin polypeptide chains.     

Autoantibody-catalyzed hydrolysis of amyloid beta peptide

We describe IgM class human autoantibodies that hydrolyze amyloid beta peptide 1-40 (Abeta40). A monoclonal IgM from a patient with Waldenstr?m's macroglobulinemia hydrolyzed Abeta40 at the Lys-28-Gly-29 bond and Lys-16-Ala-17 bonds. The catalytic activity was inhibited stoichiometrically by an electrophilic serine protease inhibitor. Treatment with the catalytic IgM blocked the aggregation and toxicity of Abeta40 in neuronal cell cultures. IgMs purified from the sera of patients with Alzheimer disease (AD) hydrolyzed Abeta40 at rates superior to IgMs from age-matched humans without dementia. IgMs from non-elderly humans expressed the least catalytic activity. The reaction rate was sufficient to afford appreciable degradation at physiological Abeta and IgM concentrations found in peripheral circulation. Increased Abeta concentrations in the AD brain are thought to induce neurodegenerative effects. Peripheral administration of Abeta binding antibodies has been suggested as a potential treatment of AD. Our results suggest that catalytic IgM autoantibodies can help clear Abeta, and they open the possibility of using catalytic Abs for AD immunotherapy.