Enzymic detennination

Most reactions in cells are carried out by enzymes [1], In many instances the rates of enzyme-catalysed reactions are enhanced by a factor of a million. A significantly large fraction of all known enzymes are proteins which are made from twenty naturally occurring amino acids. The amino acids are linked by peptide bonds to fonn polypeptide chains. The primary sequence of a protein specifies the linear order in which the amino acids are linked. To carry out the catalytic activity the linear sequence has to fold to a well defined tliree-dimensional (3D) stmcture. In cells only a relatively small fraction of proteins require assistance from chaperones (helper proteins) [2]. Even in the complicated cellular environment most proteins fold spontaneously upon synthesis. The detennination of the 3D folded stmcture from the one-dimensional primary sequence is the most popular protein folding problem. 

If a catalyst is to work well in solution, it (and tire reactants) must be sufficiently soluble and stable. Most polar catalysts (e.g., acids and bases) are used in water and most organometallic catalysts (compounds of metals witli organic ligands bonded to tliem) are used in organic solvents. Some enzymes function in aqueous biological solutions, witli tlieir solubilities detennined by the polar functional groups (R groups) on tlieir outer surfaces. 

Figure 3.10 — Flow manifolds for implementation of flow-through biosensors. (A) Flow injection merging-zones manifold for the bioluminescence detennination of ATP. ATP standards (30 fiL) and luciferin (30 fiL) are injected into the buffered carrier streams, each pumped at 0.7 mL/min and synchronously merged 12.5 cm downstream. Distance from merging point to immobilized enzyme coil, 2.2 cm. (Reproduced from [59] with permission of Elsevier Science Publishers). (B) Completely continuous flow manifold for the determination of NADH. (Reproduced from [71] with permission of the Royal Society of Chemistry). (C) Segmented-flow manifold for the determination of L-(+)-lactate. (Reproduced from [65] with permission of Marcel Dekker, Inc.). (D) Single-channel flow injection manifold with immobilized reagent for the detennination of glucose. (Reproduced from [77] with permission of Elsevier Science Publishers).

Cleland, W.W (1977) Detennining the chemical mechanisms of enzyme-catalyzed reactions by kinetic studies. Adv. Enzymol. 45, 273-387. 

The first total synthesis of a sialylated Lewis detenninant was reported by Palcic and her colleagues at the University of Alberta, Edmonton. In their biomimetic, combined oiganic-chemical and enzyme-catalyzed synthesis of the spacer-linked sLc pentaasaccharide 237, these authors followed the pathway of sLe biosynthesis previously discovered by Hansson and ZopC (Scheme 37). Hansson and Zopf found that microsomal preparations from the cell line S W 1116 catalyzed the transfer of a fucose residue from GDP- C fucose to position 4 of the GIcNAc residue of sialosylated lacto-A -tetraose a -D-Ncu5Acp-(2 3)-... 

Plasma coniains a variety of proteins with different functions. There are many characterized proteins whose function remains to be detennined. The biochemistry labtrratory rtmtinely measures total protein and albumin concentrations, usually in a serum specimen, and reports the globulin fraction as the difference between the first two results. Other proteins (e.g. immunoglobulins) arc measured as classes, and immunochemical methtxls arc available for measuring specific proteins and hormones. Enzymes are measured both by determining theiractivity and by immunochemical methods to assess their mass. 

Rg. 1 Relative enzyme activity at the indicated pH values. Enzyme activity was detennined with 4 mM p-NP-XylP (circle). Reaction conditions were as described in Materials and Methods ... 

The DNA sequence is detennined for any genes that are coded to produce enzymes. 

Recently, we cloned this MGDG syntha.se cDNA for the first time. Subsequently, by the functional expression in Escherichia coli, we detennined the 47 kDa protein was cucumber MGDG synthase. Here we describe our results of the cloning and functional expression of this enzyme. 

In typical enzyme catalysis, the rate-detennining step is the second step, as illusirated in Figure 14.9. The alternative, k would correspond to a reaction kinetically controlled by the formation of the enzyme-substrate complex, rather than by the product formation. 

Siegel, L.S., 1988, Human response to botulinum pentavalent (ABCDE) toxoid detennined by a neutralization test and by an enzyme-linked immunosorbent assay, J. Clin. Microbiol. 26 2351-2356. 

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