scholarly journals Cryoenzymology of trypsin. 13C-n.m.r. detection of an acyl-trypsin intermediate in the trypsin-catalysed hydrolysis of a highly specific substrate at subzero temperature

1984 ◽  
Vol 219 (2) ◽  
pp. 437-444 ◽  
Author(s):  
N E Mackenzie ◽  
J P Malthouse ◽  
A I Scott
Keyword(s):  
Subzero Temperature ◽  
Specific Substrate ◽  
Visible Absorption ◽  
Enzyme Substrate ◽  
First Time ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester

The kinetics of the trypsin-catalysed hydrolysis of the highly specific substrate N alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester were studied under cryoenzymological conditions by 13C-n.m.r. spectroscopy at pH approx. 3.0. The kinetics of this reaction are shown to be in agreement with similar studies made with the use of u.v.-visible-absorption-spectrophotometric techniques. A combination of 13C-n.m.r. spectroscopy and cryoenzymology has for the first time detected an acyl-trypsin intermediate in the hydrolysis of this highly specific substrate. The advantages and difficulties of using 13C-n.m.r. spectroscopy coupled with cryoenzymology in the detection and characterization of enzyme-substrate intermediates are discussed.

Transient-phase studies of a trypsin-catalyzed reaction

1970 ◽  
Vol 48 (12) ◽  
pp. 1793-1802 ◽  
Author(s):  
H. P. Kasserra ◽  
K. J. Laidler
Keyword(s):  
Steady State ◽  
Ph Dependence ◽  
Enzyme Concentration ◽  
Alcohol Concentration ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Steady State Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester

The stopped-flow technique has been used to study the pre-steady-state kinetics of the hydrolysis of N-carbobenzoxy-L-alanine-p-nitrophenyl ester catalyzed by trypsin. By working under conditions such that the enzyme concentration is much greater than that of the substrate, it has been possible to measure [Formula: see text] the rate constant for the conversion of the enzyme-substrate complex into the acyl enzyme. The pH dependence of [Formula: see text] reveals a pKb′ value of 6.9 for the conversion of complex into acyl enzyme, in agreement with deductions from steady-state investigations. The pH dependence of [Formula: see text] (equal to k−1 + k2)/k1) has also been determined. The results provide direct evidence for the existence of an enzyme-substrate complex for this reaction.The work has been done in various mixtures of water and isopropyl alcohol. The logarithms of the rate constants [Formula: see text] and [Formula: see text] vary linearly with 1/D, showing a decrease with increasing alcohol concentration; [Formula: see text] increases with alcohol concentration. The solvent results suggest that addition of alcohol affects the hydrophobic bonding in the protein and leads to unfolding of the enzyme.

scholarly journals Biosynthesis of anandamide and N-palmitoylethanolamine by sequential actions of phospholipase A2 and lysophospholipase D

2004 ◽  
Vol 380 (3) ◽  
pp. 749-756 ◽  
Author(s):  
Yong-Xin SUN ◽  
Kazuhito TSUBOI ◽  
Yasuo OKAMOTO ◽  
Takeharu TONAI ◽  
Makoto MURAKAMI ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Brain Homogenate ◽  
Wide Distribution ◽  
Rat Tissues ◽  
Lysophospholipase D ◽  
Pla2 Enzyme ◽  
First Time ◽  
Hydrolysis Of ◽  
The Brain

Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway. The PLA1/PLA2 activity for N-palmitoyl-PE was found in various rat tissues, with the highest activity in the stomach. This stomach enzyme was identified as group IB sPLA2 (secretory PLA2), and its product was determined as N-acyl-1-acyl-lysoPE. Recombinant group IB, IIA and V of sPLA2s were also active with N-palmitoyl-PE, whereas group X sPLA2 and cytosolic PLA2α were inactive. In addition, we found wide distribution of lysoPLD activity generating N-palmitoylethanolamine from N-palmitoyl-lysoPE in rat tissues, with higher activities in the brain and testis. Based on several lines of enzymological evidence, the lysoPLD enzyme could be distinct from the known N-acyl-PE-hydrolysing PLD. sPLA2-IB dose dependently enhanced the production of N-palmitoylethanolamine from N-palmitoyl-PE in the brain homogenate showing the lysoPLD activity. N-Arachidonoyl-PE and N-arachidonoyl-lysoPE as anandamide precursors were also good substrates of sPLA2-IB and the lysoPLD respectively. These results suggest that the sequential actions of PLA2 and lysoPLD may constitute another biosynthetic pathway for NAEs, including anandamide.

scholarly journals Assay of advanced glycation endproducts (AGEs): surveying AGEs by chromatographic assay with derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and application to N∊-carboxymethyl-lysine- and N∊-(1-carboxyethyl)lysine-modified albumin

2002 ◽  
Vol 364 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Naila AHMED ◽  
Ognian K. ARGIROV ◽  
Harjit S. MINHAS ◽  
Carlos A.A. CORDEIRO ◽  
Paul J. THORNALLEY
Keyword(s):  
Advanced Glycation Endproducts ◽  
Advanced Glycation ◽  
Structural Isomers ◽  
Glycation Endproducts ◽  
Assay Procedure ◽  
Carboxymethyl Lysine ◽  
Glycation Process ◽  
First Time ◽  
Hydrolysis Of

Glycation of proteins leads to the formation of early glycation adducts (fructosamine derivatives) and advanced glycation endproducts (AGEs). Formation of AGEs has been linked to the development of cataract, diabetic complications, uraemia, Alzheimer's disease and other disorders. AGEs are a group of compounds of diverse molecular structure and biological function. To characterize AGE-modified proteins used in studies of structural and functional effects of glycation, an assay was developed that surveys the content of early and advanced glycation adducts in proteins. The assay procedure involved enzymic hydrolysis of protein substrate, derivatization of the hydrolysate with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and HPLC of the resulting adducts with fluorimetric detection. Structural isomers of methylglyoxal-derived hydroimidazolone, glyoxal-derived hydroimidazolone, 3-deoxyglucosone-derived hydroimidazolone and Nδ-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)-ornithine (THP) were determined for the first time. AGEs with intrinsic fluorescence (argpyrimidine, pentosidine) were assayed without derivatization. Limits of detection were 2–17pmol and levels of recovery were 50–99%, depending on the analyte. The AQC assay resolved structural and epimeric isomers of methylglyoxal-derived hydroimidazolones and THP. Hydroimidazolones, THP and argpyrimidine were AGEs of short-to-intermediate stability under physiological conditions, with half-lives of 1–2weeks. Their measurement provides further insight into the glycation process. The assay was applied to the characterization of human serum albumin minimally and highly modified by N∊-carboxymethyl-lysine and N∊-(1-carboxyethyl)-lysine.

scholarly journals The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3

1982 ◽  
Vol 203 (1) ◽  
pp. 149-153 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Methyl Esters ◽  
Plasma Kallikrein ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Kinetics Of Hydrolysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.

Presteady-state kinetics of Bacillus 1,3–1,4-β-glucanase: binding and hydrolysis of a 4-methylumbelliferyl trisaccharide substrate

2001 ◽  
Vol 357 (1) ◽  
pp. 195-202
Author(s):  
Mireia ABEL ◽  
Antoni PLANAS ◽  
Ulla CHRISTENSEN
Keyword(s):  
Steady State ◽  
Rate Constant ◽  
Product Formation ◽  
Lag Phase ◽  
Wild Type ◽  
Induced Fit ◽  
Enzyme Substrate ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Presteady State Kinetics

In the present study the first stopped-flow experiments performed on Bacillus 1,3–1,4-β-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-β-cellobiosyl-β-d-glucoside to the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bound substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase leading to equilibration (for binding to E134A) or to steady state product formation (for the wild-type reaction). The lag phase, with a rate constant of the order of 100s−1, was independent of the substrate concentration; apparently an induced-fit mechanism governs the formation of enzyme–substrate complexes. The concentration dependencies of the observed rate constant of the second presteady-state phase were analysed according to a number of reaction models. For the reaction of the wild-type enzyme, it is shown that the fast product formation observed before steady state is not due to a rate-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change of the productive ES complex into a form that binds a second substrate molecule in a non-productive mode.

SYNTHESIS AND CHARACTERIZATION OF DYE (PHENOSAFRANINE) SENSITIZED FLOWER-TYPE ZnO NANORODS

2010 ◽  
Vol 24 (10) ◽  
pp. 1289-1298 ◽  
Author(s):  
N. RAJKUMAR ◽  
R. N. MARIAMMAL ◽  
K. RAMACHANDRAN
Keyword(s):  
Zno Nanorods ◽  
Synthesis And Characterization ◽  
Absorption Analysis ◽  
Photoluminescence Spectra ◽  
Visible Absorption ◽  
Dye Sensitized ◽  
Temperature Solution ◽  
Flower Type ◽  
First Time

Flower-type ZnO nanorods were synthesized by two-step low temperature solution growth and dye-sensitized with phenosafranine for the first time. The scanning electron microscope result shows that the diameter and the length of a single rod of the flower-type nanostructures depend on the method of synthesis. Optical absorption analysis shows a visible absorption at 517 nm, which was otherwise absent in nano ZnO . The photoluminescence spectra of ZnO and dye-sensitized ZnO nano flowers were also analyzed.

scholarly journals Characterization of the CDP-2-Glycerol Biosynthetic Pathway in Streptococcus pneumoniae

2010 ◽  
Vol 192 (20) ◽  
pp. 5506-5514 ◽  
Author(s):  
Quan Wang ◽  
Yanli Xu ◽  
Andrei V. Perepelov ◽  
Wei Xiong ◽  
Dongmei Wei ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Streptococcus Pneumoniae ◽  
Biosynthetic Pathway ◽  
Resonance Spectroscopy ◽  
Ionization Mass ◽  
Enzyme Substrate ◽  
Effects Of Temperature ◽  
First Time ◽  
Novel Products

ABSTRACT Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtp1, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzyme-substrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase that catalyzes the conversion of 1,3-dihydroxyacetone to glycerol, Gtp3 was identified as a glycerol-2-phosphotransferase that catalyzes the conversion of glycerol to glycerol-2-phosphate, and Gtp2 was identified as a cytidylyltransferase that transfers CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtp1 and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically; this pathway provides a method to enzymatically synthesize this compound.

scholarly journals Evaluation of the extent of the binding site in human tissue kallikrein by synthetic substrates with sequences of human kininogen fragments

1995 ◽  
Vol 312 (1) ◽  
pp. 233-238 ◽  
Author(s):  
E Del Nery ◽  
J R Chagas ◽  
M A Juliano ◽  
E S Prado ◽  
L Juliano
Keyword(s):  
Human Tissue ◽  
Kinetic Data ◽  
Time Course ◽  
Tissue Kallikrein ◽  
Hydrolysis Time ◽  
Porcine Tissue ◽  
Enzyme Substrate ◽  
Substrate Side ◽  
Kinetics Of ◽  
Hydrolysis Of

We have synthesized internally quenched peptides spanning the Met379-Lys380 or Arg389-Ser390 bonds of human kininogen (hkng) that flank lysyl-bradykinin and have studied the kinetics of their hydrolysis by human tissue kallikrein. The kinetic data for the hydrolysis of the Met-Lys bond in substrates with an N-terminal extension showed that interactions up to position residue P10 contribute to the efficiency of cleavage. In contrast, there were no significant variations in the kinetic data for the hydrolysis of substrates with C-terminal extensions at sites P′4 to P′11. A similar pattern was observed for the cleavage of substrates containing an Arg-Ser bond because substrates extended up to residue P6 were hydrolysed with the highest kcat/Km values in the series, whereas those extended to P′11 on the C-terminal side had a lower susceptibility to hydrolysis. Time-course studies of hydrolysis by human and porcine tissue kallikreins of a Leu373 to Ile393 human kininogen fragment containing omicron-aminobenzoic acid (Abz) at the N-terminus and an amidated C-terminal carboxyl group Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg- Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Ile-NH2 (Abz-[Leu373-Ile393]-hkng-NH2) indicated that the cleavage of Met-Lys and Arg-Ser bonds in the same molecule occurs via the formation of independent enzyme-substrate complexes. The hydrolysis of Abz-F-R-S-S-R-Q-EDDnp [where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] and Abz-M-I-S-L-M-K-R-P-Q-EDDnp by human tissue kallikrein had maximal kcat/Km values at pH 9-9.5 for both substrates. The pH-dependent variations in this kinetic parameter were almost exclusively due to variations in kcat. A significant decrease in kcat/Km values was observed for the hydrolysis of Arg-Ser and Met-Lys bonds in the presence of 0.1 M NaCl. Because this effect was closely related to an increase in Km, it is likely that sodium competes with the positive charges of the substrate side chains for the same enzyme subsites.

scholarly journals Spectroscopic studies of the type 2 and type 3 copper centres in the mercury derivative of laccase

1989 ◽  
Vol 263 (2) ◽  
pp. 425-429 ◽  
Author(s):  
R Tamilarasan ◽  
D R McMillin
Keyword(s):  
Structural Transition ◽  
Spectroscopic Studies ◽  
Ligand Field ◽  
Visible Absorption ◽  
Mercury Derivative ◽  
Spectral Changes ◽  
Type 3 ◽  
First Time ◽  
Kinetics Of

U.v.-visible-absorption and e.p.r. spectroscopy were used to study the type 2 and type 3 copper centres in the mercury derivative of laccase. After treatment with peroxide the mercury derivative of laccase exhibits a fully developed absorption band at 330 nm (delta epsilon = 2900 +/- 100 M-1.cm-1, which is characteristic of type 3 copper in the oxidized state. In addition, there is a weak ligand-field absorption at 740 nm (epsilon = 380 +/- 30 M-1.cm-1), which can be assigned to the type 3 pair. Because the e.p.r. spectrum of the type 2 copper is well resolved in the case of the mercury derivative of laccase, for the first time we have been able to observe spectroscopic evidence for a pH-dependent structural transition that has been invoked to explain the kinetics of enzyme reduction [Andréasson & Reinhammar (1979) Biochim. Biophys. Acta 568, 145-156]. According to the e.p.r. data the pKa lies in the range 6-7, and comparisons with a model compound show that the spectral changes can plausibly be interpreted in terms of the deprotonation of a water molecule in the co-ordination sphere of the type 2 copper.

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