Amylases substrates

Fig. 1. Inhibition of porcine pancreatic a-amylase. Substrates, an inhibitor, and their binding orientations in the active site are shown schematically. The arrows denote the catalytic site in each case, (a) The small substrate, G2PNP [17400-77-0] (3) (b) the large substrate, G OH [13532-61 -1] (4) and (c) the inhibitor, 4-phenyl imidazole (5) and the substrate G2PNP (3) in the binding orientation for noncompetitive inhibition. The binding orientation of G2PNP...

Zhang Z., Seitz W.R., O Connell K., Amylase substrate based on fluorescence energy... 

Z. Zhang, W. R. Seitz, and K. O Connel, Amylase substrate based on fluorescence energy transfer, Anal. Chim. Acta 236, 251-256 (1988). 

Pluorogenlc malto-ollgosaccharldes with 6-deoxy-6-(2-pyrldyl)-amlno-substltuted non-reducing terminal units and their corresponding alditol derivatives (from NaBH reduction), required as o-amylase substrates, have been synthesized by reductive cimination (NaBH CN) of partially 6-aldehydo-amylose with 2-amlno-... 

A number of dye-labelled amylase substrates have been introduced in recent years by several companies. A coloured dye is coupled to starch substrate. Hydrolysis of the substrate by the enzyme results in the formation of water-soluble dye fragments which can be separated from the unhydrolysed substrate by centrifugation or filtration. 

An example of a direct spectrophotometrical assay is the use of synthetic peptide -nitroanilide substrates to determine protease activity. The /)-nitroani1ine group Hberated from the substrates by the protease can be determined spectrophotometricaHy at 410 nm. An example of an indirect (coupled) spectrophotometric assay is the determination of a-amylase using -nitrophenyLmaltoheptaoside. Initially, the substrate is cleaved by the a-amylase and subsequentiy one of the reaction products, -nitrophenyLmaltotrioside, is cleaved by a-glucosidase, hberating -nitrophenyl, a chromophore... 

By the end of the nineteenth century a more descriptive system was in use. The suffix -ase was appended to the name of the substrate involved in the reaction, eg, amylase, ceUulase, protease, Hpase, urease, etc. Names that reflected the function of the enzyme with the suffix -ase were also used, eg, invertase, transferase, isomerase, oxidase. 

Unlike many of the catalysts that chemists use in the laboratory, enzymes are usually specific in their action. Often, in tact, an enzyme will catalyze only a single reaction of a single compound, called the enzyme s substrate. For example, the enzyme amylase, found in the human digestive tract, catalyzes only the hydrolysis of starch to yield glucose cellulose and other polysaccharides are untouched by amylase. 

Different enzymes have different specificities. Some, such as amylase, are specific for a single substrate, but others operate on a range of substrates. Papain, for instance, a globular protein of 212 amino acids isolated from papaya fruit, catalyzes the hydrolysis of many kinds of peptide bonds. In fact, it s this ability to hydrolyze peptide bonds that makes papain useful as a meat tenderizer and a cleaner for contact lenses. 

Fig. 24. Kinetics of add inactivation of a-amylase (Bac. subtilis) in solution (/, 2) and immobilized on Biocarb (3) 1) pH 2 2) and 3) pH 4. A/A0 is the value of relative enzymatic activity (compared to the initial activity A0 before inactivation), substrate — starch...

A single hydrolase is usually inadequate for the degradation of a carrier, but most hydrolases have unspecific activities, i.e., they split the chains of polymers that are not their typical substrates. For example, chitosan is susceptible to lipases, pectinases, amylases among others [257-260]. 

Crystal-structure analysis of Taka amylase A gave similar results, in that it showed that it had an extended cleft which could accommodate six, or possibly seven, a-( 1 — 4)-linked glucose units and two oppositely placed acidic amino acids (Asp-206 and Glu-230) which could interact with the bound substrate similarly to Asp-52 and Glu-35 in lysozyme. 

Hehre and coworkers showed that beta amylase from sweet potatoes, an inverting, a-specific exo-(l 4)-glucanase, catalyzes the hydrolysis of jS-maltosyl fluoride with complex kinetics which indicated the participation of two substrate molecules in the release of fluoride ion. Furthermore, the reaction was strongly accelerated by the addition of methyl ) -maltoside. Hydrolysis of a-maltosyl fluoride, on the other hand, obeyed Michaelis-Menten kinetics. The main product with both a- and yj-maltosyl fluoride was )S-maltose. The results with )3-maltosyl fluoride were interpreted by the assumption of a glycosylation reaction preceding hydrolysis by which a malto-tetraoside is formed by the replacement of fluoride ion by a second substrate molecule or added methyl -maltoside (see Scheme 5). 

In the late 1960 s a new series of methods was introduced for the determination of amylase, involving the use of a starch-dye complex. Dyes such as Renazol brilliant blue (68) Reactive Red 2B (69) (used in the substrate Dy-Amyl, General Diagnostic Division, Warner-Chilcott Laboratories), Cibachrom Blue (70)... 

In the Phadebas TM amylase test (72) (Pharmacia Labs) the substrate was a water insoluble cross-TTnked blue starch in tablet form which also contains some inert ingredients, sodium and potassium phosphate buffer salts and sodium chloride. This polymer was hydrolyzed by amylase into water soluble blue starch fragments. After centrifugation the absorbance of the blue supernatant was proportional to the activity of amylase present in the test samples. The day to day variation on a quality control serum had a coefficient of variation of 2.7% based on 30 days of data in our laboratory. The method is simple, reproducible and uses microquantities of serum. 

Amylases, peptidases and deoxyribonuclease mobilize many nutrients that are released from lysed cells. They also deerease the viseosity of fluids present at the lesion by depolymerization of their biopolymer substrates. 

The magnitude of inhibition of polygalacturonase was found to be dependent on preincubation of inhibitor with the enzyme. Similar observations have been reported for other enzyme inhibitors (Shivaraj and Pattabiraman, 1980 Sharma and Pattabiraman, 1980 Padmanabhan and Shrasti, 1990). However, preincubation of the inhibitor with substrate did not show any effect on inhibitor activity. In contrast, Shivaraj and Pattabiraman (1980) and Buonocore et al. (1977), have observed inactivation of amylase inhibitor activity on pretreatment with starch. 

The diastase activity was traditionally determined according to the Schade method in the earlier years (Schade et al., 1958). One unit of diastase activity (or more specifically, a-amylase), DN, is defined as that amoimt of enz)nne that converts 0.01 g of starch to the prescribed endpoint in 1 h at 37 °C under the experimental conditions. In this assay, a standard solution of starch, which reacts with iodine to produce a color solution, is used as a substrate for honey enzymes under the standard conditions (Rendleman, 2003). A recently developed procedure uses an insoluble, dyed starch substrate (Persano Oddo and Pulcini, 1999). As this substrate is hydrolyzed by ot-amylase, soluble dyed starch fragments are released into solution. After reaction termination and insoluble substrate removal by centrifugation, absorbance of the supernatant solution (at 620 nm) is measured. The absorbance is proportional to the diastase activity. This procedure has been widely adopted in the honey industry due to the convenience of a commercially available substrate and the simple assay format. 

Some of the pancreatic enzymes in the lumen include pancreatic amylase, pancreatic lipase, elastase, trypsin, a-chymotrypsin, and carboxypeptidase A. For example, the aspirin derivatives aspirin phenylalanine ethyl ester, aspirin phenyllactic ethyl ester, and aspirin phenylalanine amide have been studied as substrates for carboxypeptidase A [67,68], with the phenylalanine ethyl ester derivative proving to be the best substrate. This study indicated that the carboxypeptidase A may serve as a reconversion site for many drug derivatives. 

Payre, N., Cottaz, S. and Driguez, H. (1995). Chemoenzymatic synthesis of a modified pentasaccharide as a specific substrate for a sensitive assay of alpha-amylase by fluorescence quenching. Angew. Chem. Int. Ed. Engl. 34, 1239-1241. 

---