Amylase multiple attack

The investigations carried out by Professor French and his students were based on sound experimental approaches and on intuitive theoretical considerations. The latter often resulted in new experiments for testing a hypothesis. On the basis of theoretical considerations, Professor French proposed a model for the structure of the amylopectin molecule, and the distribution of the linear chains in this molecule. This model was tested by utilizing enzymes that selectively cleave the linear chains, and the results substantiated the theoretical deductions. He proposed a theory on the nature and types of reactions occurring in the formation of the enzyme - starch complex during the hydrolysis of starch by amylases. In this theory, the idea of multiple attack per single encounter of enzyme with substrate was advanced. The theory has been supported by results from several types of experiments on the hydrolysis of starch with human salivary and porcine pancreatic amylases. The rates of formation of products, and the nature of the products of the action of amylase on starch, were determined at reaction conditions of unfavorable pH, elevated temperatures, and increased viscosity. The nature of the products was found to be dramatically affected by the conditions utilized for the enzymic hydrolysis, and could be accounted for by the theory of the multiple attack per single encounter of substrate and enzyme. 

A. oryzae a-amylase has about the same degree of multiple attack as human salivary a-amylase, i.e. an average of three hydrolytic cleavages per chain encounter. A. oryzae a-amylase is the only common a-amylase known to hydrolyze the cyclic a-(l- 4)-l inked maltodextrins to any extent.25,26 A kinetic analysis revealed that the catalytic constant, k2, increased significantly from 3.3 to 270 to 3270min-1 for cyclomaltohexaose, cyclomaltoheptaose and cyclomalto-octaose, respectively, while the Km was 4.7 M, 10.2 M and 2.4 M, respectively.26... 

Mammalian aipha-amylases probably attack large substrates by a multiple-attack mechanism, but it seems unlikely that any of the other well studied types of alpha-amylase act in this way. 

The action of B. subtilis alpha-amylase is probably random on the internal bonds of amylose, but non-random attack occurs near chain ends. Further calculations indicate that little, if any, multiple attack occurs. Few results have been obtained for the enzyme from A. oryzae, but it is believed that multiple attack does not occur. ... 

DP /DP = 1.5) is employed. It may be seen that (a) with natural amylose, DP is invariant during the hydrolysis, (b) in the case of the amylose fraction, an initial decrease in DP, is followed by invariance due to the regeneration of the exponential function, and (c) the value of DP for the synthetic amylose decreases constantly during the hydrolysis. The evidence to date, therefore, suggests that heta-amylase action is not single-chain, but either multi-chain or limited multiple attack—the experimental results cannot distinguish between the two. 

A third mechanism, proposed by French,involves multiple attack, in which the enzyme remains associated with a given substrate molecule long enough to remove several maltose residues before attacking another amylose molecule. With amylose of DP 44, sweet-potato /3-amylase removed about four maltose residues per effective encounter. i The multiple-attack mechanism is, in fact, intermediate between the single-chain and multi-chain patterns. In agreement with this view, Whelan and Bailey found that the action of /3-amylase on maltosaccharides of DP 6 and 7 and on amylose of DP 49 was intermediate between single-chain and multichain, but varied with the pH and temperature of the experiments. 

The mechanism by which multiple attack takes place is not at all clear, although the finding that pig pancreatic amylase has two apparently independent binding sites for substrates and forms multimolecular enzyme-substrate complexes (127) suggests a way in which enzyme and substrate could be constrained to continue the association necessary for multiple attack. 

Theoretical models for the action of a-amylase on amylose have indicated that multiple attack is of little or no significance in the action patterns of a-amylases, with the exception of those from the pancreas. Six a-limit dextrins containing a... 

Studies of the hydrolyses of cyclohexa-, cyclohepta-, and cyclo-octa-amyloses by Taka-amylase A (one of the a-amylases from Aspergillus oryzae) indicated that each cycloamylose binds to the same active site of the enzyme. Since there is little difference in the respective AG, Af/, and AS values for enzymic hydrolysis of these cycloamyloses, their binding modes appear to be similar. The extent of multiple attack on the cycloamyloses was not affected by temperature. A 4-phenylazobenzoyl derivative of Taka-amylase A has been used to investigate the active site of the enzyme. A. oryzae a-amylase has a synergistic effect on the action of the glucoamylase from A. awamori var. kawachi ... 

Several models have been proposed to account for the non-random distribution of oligosaccharides formed when polysaccharides are hydrolysed by a-amylase. The preferred-attack model assumes that the probability of bond-cleavage depends on the position of the bond in the chain the repetitive (or multiple-attack) model assumes that a-amylase can form a cage-like complex with the substrate and attack it several times during a single encounter the multiple-enzyme (or dual-site) model assumes that the substrate is hydrolysed by the combined actions of exo- and en /o-enzymes. The effects of pH, inhibitors, and the chain length of the substrates have been studied in an attempt to decide which of the three models best fits the action of a-amylase. The effects of these variables on either the distribution of products or the action pattern of the enzyme were incorporated into the models, which were then used to interpret experimental data obtained with porcine pancreatic a-amylase. 

Ishikawa, K, Nakatani, H., Katsuya, Y. Fukazawa, C. (2007). Kinetic and Structural Analysis of Enzyme Sliding on a Substrate Multiple Attack in P-Amylase. Biochemistry, Vol. 46, No.3, pp. 792-798... 

---