Multichain enzymes

Whelan and Bailey were also able to clarify the polymerization mechanism of the enzymatic polymerization with phosphorylase [124], Their results showed that the polymerization follows a multichain scheme in contrast to a single-chain scheme that was also proposed by some authors. In the multichain polymerization scheme, the enzyme-substrate complex dissociates after every addition step, whereas in the single-chain scheme each enzyme continuously increases the length of a single primer chain without dissociation. 

If enzyme inactivation is not due to covalent changes in structure, the native active structure can be reformed in immobilized enzymes by refolding from a random coil state (22,27). In fact, if they are attached to the matrix by multiple points, even thermally inactivated multichain enzymes can be reactivated (27). Although such regeneration steps apparently have not been used commercially, their use should be considered in certain cases, especially since in many cases the inactivation may result from adsorption onto the enzyme matrix of components in the process stream. For example, we have found that immobilized sulfhydryl oxidase activity can be regenerated numerous times following treatment of UHT milk by washing with 4 M urea (28). 

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. 

Although the synthesis of amylose by P-enzyme proceeds by a multichain mechanism, the reverse reaction shows some degree of single-chain action. The final products of phosphorolysis are a-D-glucosyl phosphate and maltopentaose. 

Indications of a structural role for metals in a multichain enzyme were actually noted nearly a decade ago e.g., the removal of zinc from yeast alcohol dehydrogenase with chelating agents resulted in dissociation of the protein into subunits (6). During the intervening years, additional investigations have suggested that this structural role for metals may be rather common and, indeed, have important implications to enzymatic function. 

Today, for the purpose of illustrating these points, we wish to review selected segments of work performed in our laboratory on two multichain enzymes—alkaline phosphatase from . coli and equine liver alcohol dehydrogenase—in which intrinsic metal atoms appear to play both functional and structural roles. Thereafter, we hope to discuss data suggesting that metals may be important also to the structure of certain single-chain proteins and to the general phenomenon of stabilization of protein structure. 

Synthesis of the skeletal chains must proceed, as do all enzyme-controlled polymerization reactions, either by a single or a multichain reaction mechanism, through a series of "growing cycles in which the... 

The enzyme is always bound to the polymer chain in this one-chain mechanism. Multichain mechanisms are much more common. In these, the enzyme disengages after every linking step and thus wanders from chain to chain (see also polycondensation. Chapter 17). In the multichain mechanism, the enzyme and the substrate form an enzyme-substrate complex ES in the first step ... 

The preceding derivations relate to a multichain mechanism with one catalytically active group per enzyme molecule. If the enzyme molecule has N equally and quite independently active groups, then Equation (19-54) is modified to... 

Covalent modHication of enzymes, enzyme modulation, enzyme interconversion Oligomeric (i.e. multichain) enzymes may exist in two or more forms, which are interconvertible by enzyme-catalysed covalent modifications, and which differ in their catalytic properties, e.g. activity, substrate affinity and dependence on effectors. Usually the difference in activity is such that one form is active and the other inactive. The activities of the conversion enzymes are in turn regulated by other enzymes, metabolites and/ or effectors. Covalent modifications are therefore important in physiological regulation, in addition to Allostery (see). Whereas allostery provides fine adjustment of metablic rates, C provides an on/off switching of cellular functions, which is very sensitive to environmental influences. 

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