Single enzyme catalysis

The idea to analyze the catalysis by a single enzyme molecule was an obvious step. This was first demonstrated by Peter Lu and Sunney Xie [38]. They were able to show intensity fluctuation of the flavin cofactor of cholesterol oxidase using confocal single molecule detection. Our idea of single enzyme catalysis was to follow the turnover of a substrate into the product by catalysis of a single enz une molecule. We chose horse radish peroxidase and a non-fluorescence substrate dihydro-rhodamine which is turned over in the fluorescent product... 

We have interpreted the observation of the stretched exponential behavior as being caused by the manifold of transition rates involved in the substrate turnover. This has been observed for all cases where the single enzyme catalysis has been studied by the product turnover [43-45] (Fig. 4.19). 

First-order rate constants are used to describe reactions of the type A — B. In the simple mechanism for enzyme catalysis, the reactions leading away from ES in both directions are of this type. The velocity of ES disappearance by any single pathway (such as the ones labeled k2 and k3) depends on the fraction of ES molecules that have sufficient energy to get across the specific activation barrier (hump) and decompose along a specific route. ES gets this energy from collision with solvent and from thermal motions in ES itself. The velocity of a first-order reaction depends linearly on the amount of ES left at any time. Since velocity has units of molar per minute (M/min) and ES has units of molar (M), the little k (first-order rate constant) must have units of reciprocal minutes (1/min, or min ). Since only one molecule of ES is involved in the reaction, this case is called first-order kinetics. The velocity depends on the substrate concentration raised to the first power (v = /c[A]). 

EFFECT OF ADDITIONAL CENTRAL COMPLEX SPECIES ON THE GENERAL FORM OF THE STEADY STATE RATE EOUATION. Up to now, we have actually considered a chemically unrealistic model for enzyme catalysis in that we have assumed that a single enzyme-bound species, namely EX, accounts for the catalytic process. We now treat a more reasonable representation of the kinetic mechanism... 

Johnson and Fierke Hammes have presented detailed accounts of how rapid reaction techniques allow one to analyze enzymic catalysis in terms of pre-steady-state events, single-turnover kinetics, substrate channeling, internal equilibria, and kinetic partitioning. See Chemical Kinetics Stopped-Flow Techniques... 

The considerable detail to which we now can understand enzyme catalysis is well illustrated by what is known about the action of carboxypeptidase A. This enzyme (Section 25-7B and Table 25-3) is one of the digestive enzymes of the pancreas that specifically hydrolyze peptide bonds at the C-terminal end. Both the amino-acid sequence and the three-dimensional structure of carboxypeptidase A are known. The enzyme is a single chain of 307 amino-acid residues. The chain has regions where it is associated as an a helix and others where it is associated as a /3-pIeated sheet. The prosthetic group is a zinc ion bound to three specific amino acids and one water molecule near the surface of the molecule. The amino acids bound to zinc are His 69, His 196, and Glu 72 the numbering refers to the position of the amino acid along the chain, with the amino acid at the /V-terminus being number l. The zinc ion is essential for the activity of the enzyme and is implicated, therefore, as part of the active site. 

Escherichia coli have also developed an elegant method to control enzyme catalysis that occurs by covalent modification of each subunit. In this latter reaction a single tyrosyl residue per subunit is adenylylated to produce a stable 5 -adenylyl-O-tyrosyl derivative. Recent NMR and fluorescence data will be reviewed concerning the nature of this adenylyl site and its spatial relationship to the metal ions at the catalytic site. The enzymes responsible for the covalent adenylylation reaction comprise a cascade system for amplifying the activation or inactivation of glutamine synthetase molecules (81). 

Structural studies of the oxy-Cope catalytic antibody system reinforce the idea that conformational dynamics of both protein and substrate are intimately intertwined with enzyme catalysis, and consideration of these dynamics is essential for complete understanding of biologically catalyzed reactions. Indeed, recent single molecule kinetic studies of enzyme-catalyzed reactions also suggest that different conformations of proteins are associated with different catalytic rates (Xie and Lu, 1999). In addition, a number of enzymes are known to undergo conformational changes on binding of substrate (Koshland, 1987) that lead to enhanced catalysis two examples are hexokinase (Anderson and Steitz, 1975 Dela-Fuente and Sols, 1970) and triosephosphate isomerase (Knowles, 1991). 

For other enzymes activity has been detected in some cases at 0.1 h or, rarely, at a lower hydration level. Most of the enzymes studied show onset of activity between 0.1 and 0.2 h, but there is not enough information to arrive at a consensus value. There appears to be no single hydration level that is critical for enzyme catalysis. Perhaps it is to be expected that different mechanisms should be associated with different roles of solvent. 

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