Enzymology: substrate

Perhaps the biggest impact on the practical utilization of enzymes has been the development of nonaqueous enzymology (11,16,33,35). The use of enzymes in nonaqueous media gready expands the scope of suitable transformations, simplifies thek use, and enhances stabiUty. It also provides an easy means of regulation of the substrate specificity and regio- and enantioselectivity of enzymes by changing the reaction medium. 

Singleton, V. L., Orthofer, R., and Lamuela-Raventos, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. In "Methods in Enzymology, Oxidant and Antioxidants (Part A)", (L. Packer, Ed.), vol. 299 pp. 152-178. Academic Press, San Diego, CA. 

Enzymology of the Formation of Hydroxyacyl-CoA Thioesters as Substrates for PHA Synthases... 

The mode of action of enzymes can be found in detail in many biochemistry and enzymology textbooks31"33. The mechanisms of enzyme-catalyzed reactions are complex and all have several steps. The more generally written scheme involves a single substrate and a single intermediate ... 

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) is another recently introduced technique for quantification of proteins, and to date has most often been applied to clinical enzymology.60 The product conjugates of the enzymatic reaction between the synthetic substrate and targeted enzyme are captured by immobilized affinity reagents, purified, released into solution, and analyzed by ESI-MS. 

Anyhow, our study has demonstrated the benefit of "strained" polymeric catalyst-substrate complexes, a phenomenon well-known in enzymology (26) and once indicated by the term "entatic state" (16). 

The study of individual NRPS domain structures provides important information regarding the specificity and enzymology of individual steps in NRP biosynthesis. However, structural analysis of larger NRPS constructs is necessary to gain insight into aspects related to domain/domain interactions and the overall structure of the synthetase machinery. This information is particularly important for understanding the details of substrate trafficking and will assist efforts toward the rational manipulation of NRPSs. 

Selected entries from Methods in Enzymology [vol, page(s)] Acetate assay with, 3, 269 activation, 44, 889 activity assay, 44, 893, 894 alternative substrates, 87, 11 bridge-to-nonbridge transfer, 87, 19-20, 226, 232 chiral phosphoryl-ATP, 87, 211, 258, 300 cold denaturation, 63, 9 cysteine residues, 44, 887-889 equilibrium constant, 63, 5 exchange properties, 64, 9, 39, 87,... 

Selected entries from Methods in Enzymology [vol, page(s)] Acetylthiocholine as substrate, 251, 101-102 assay by ESR, 251, 102-105 inhibitors, 251, 103 modification by symmetrical disulfide radical, 251, 100 thioester substrate, 248, 16 transition state and multisubstrate analogues, 249, 305 enzyme receptor, similarity to collagen, 245, 3. 

Selected entries from Methods in Enzymology [vol, page(s)] Assay, 1, 611 3, 935-938 63, 33 separation by HPLC, 72, 45 extraction from tissues, 13, 439 formation of, 1, 486, 518, 585 5, 466 free energy of hydrolysis, 1, 694 substrate for the following enzymes [acetyl-coenzyme A acyl carrier protein transacylase, 14, 50 acetyl-coenzyme A carboxylase, 14, 3, 9 acetyl-coenzyme A synthetase, 13, 375 N-acetyltransferase, 17B, 805 aminoacetone... 

Selected entries from Methods in Enzymology [vol, page(s)] Equilibrium isotope exchange study of kinetic mechanism, 249, 466 site-directed mutagenesis of Escherichia coli enzyme, 249, 93 positional isotope exchange studies, 249, 423 product inhibition studies of three substrates three products reactions, 249, 207-208. 

Selected entries from Methods in Enzymology [vol, page(s)] Active site, structure, 241, 214 catalytic mechanism, 241, 223-224 crystal structure, 241, 214, 216 comparative studies with HIV protease [catalytic properties, 241, 205-224 evolutionary relationships, 241, 196-197 screening for HIV-1 protease inhibitors, 241, 318-321 structure, 241, 254-257, 280 substrate specificity, 241, 255, 283]. 

Selected entries from Methods in Enzymology [vol, page(s)] Design, 178, 551 immunoassay, 178, 542 production, 178, 531 purification, 178, 543 substrates and enzymatic assay, 178, 544 derivatization with spectroscopic probe, 178, 567 ester cleavage assays, 178, 565 fluorescence quenching binding assay, 178,... 

CATALYSIS. Any condition promoting formation will tend to speed up the reaction rate, and catalysts are thought to accomplish rate enhancement chiefly by stabilizing the transition state. Shown in Fig. 8 is an enzyme-catalyzed process in which reactant S (more commonly called substrate in enzymology) combines with enzyme to form an enzyme-substrate complex. This complex leads to formation of the transition state complex EX which may proceed to form enzyme-product complex. The catalytic reaction cycle is then completed by the release of product P, whereupon the uncombined enzyme returns to its original state. 

Selected entries from Methods in Enzymology [vol, page(s)] Active site titration [by 2-hydroxy-5-nitro-a-toluenesulfonic acid sultone, 19, 6-14 by p-nitrophenyl ester substrates, 19, 14-20 by rapidly reversible, covalently bound substrates, 19, 14-20 by slowly reversible, covalently bound inhibitors, 19, 6-14] assay,... 

Selected entries from Methods in Enzymology [vol, page(s)] Dilution of enzyme samples, 63, 10 lipolysis substrate effect, 64, 361, 362 dilution jump kinetic assay, 74, 14-19, 28 dilution method [for dissociation equilibria, 61, 65-96 continuous dilution cuvette, 61, 78-96 data analysis, 61, 74, 75 equations, 61, 70-74 errors, 61, 76-78 experimental procedures, 61, 69, 70 merits, 61, 75, 76 theory, 61, 68, 69... 

Selected entries from Methods in Enzymology [vol, page(s)] Application in fluorescence, 240, 734, 736, 757 convolution, 240, 490-491 in NMR [discrete transform, 239, 319-322 inverse transform, 239, 208, 259 multinuclear multidimensional NMR, 239, 71-73 shift theorem, 239, 210 time-domain shape functions, 239, 208-209] FT infrared spectroscopy [iron-coordinated CO, in difference spectrum of photolyzed carbonmonoxymyo-globin, 232, 186-187 for fatty acyl ester determination in small cell samples, 233, 311-313 myoglobin conformational substrates, 232, 186-187]. 

Selected entries from Methods in Enzymology [vol, page(s)] Enzyme-substrate complex formation, 64, 53 data analysis, 64, 56, 57 extensions of technique, 64, 57-59 as evidence for occurrence of intermediate, 64, 47-59 kinetic equation, 64, 49-52 limitations, 64, 57-59 mixing procedure, 64, 53-56 reaction condition, 64, 56, 57 termination, 64, 56, 57. 

A unit of enzyme catalytic activity equal to the conversion of one mole of substrate per second in a specified assay system. The katal (kat) is more commonly used in clinical enzymology. One unit of enzyme activity (i.e., one micromole per minute) corresponds to 16.67 nkat. 

Periodic acid oxidation has proved to be a very useful tool in enzymology since a wide variety of biochemicals contain hydroxyl groups on adjacent carbon atoms. For example, periodate-oxidized ATP (also called adenosine 5 -triphosphate 2, 3 -dialdehyde) has often been used as an alternative substrate or an irreversible inhibitor for a wide variety of ATP-utilizing enzymes. This compound, and many others, are now commercially available, even though they are readily synthesized e.g., periodic acid oxidized ADP, AMP, adenosine, P, P -di(adenosine-5 )pentaphosphate, P, P -di(adenosine-5 )tetraphos-phate, GTP, GDP, GMP, guanosine, CTP, CDP, CMP, etc. In the case of the nucleosides, commercial sources also can supply the dialcohol form of the nucleoside i.e., the nucleoside has first been oxidized with periodic acid and then reduced to the dialcohol with borohydride. 

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