Enzyme Substrate Interactions

The simplest way to prepare a biocatalyst for use in organic solvents and, at the same time, to adjust key parameters, such as pH, is its lyophilization or precipitation from aqueous solutions. These preparations, however, can undergo substrate diffusion limitations or prevent enzyme-substrate interaction because of protein-protein stacking. Enzyme lyophilization in the presence of lyoprotectants (polyethylene glycol, various sugars), ligands, and salts have often yielded preparations that are markedly more active than those obtained in the absence of additives [19]. Besides that, the addition of these ligands can also affect enzyme selectivity as follows. 

The successful use of these X-ray crysallographic techniques in studying the enzyme-substrate interactions of lysozyme (21) and chymotrypsin (22) has recently been reviewed by Blow and Steitz (16) and Blow (23). To date, however, these methods have had only limited application, since the detailed structures of only about ten enzymes have been elucidated by X-ray diffraction... 

Early in the last century, Emil Fischer compared the highly specific fit between enzymes and their substrates to that of a lock and its key. While the lock and key model accounted for the exquisite specificity of enzyme-substrate interactions, the imphed rigidity of the... 

Tanizawa, K., and Kanaoka, Y. Design of Biospecific Compounds which Simulate Enzyme-Substrate Interaction. 136, 81-107 (1986). 

In kinetic studies of enzymatic reactions, rate data are usually tested to determine if the reaction follows the Michaelis-Menten model of enzyme-substrate interaction. Weetall and Havewala [Biotechnol. and Bioeng. Symposium 3 (241), 1972] have studied the production of dextrose from cornstarch using conventional... 

More recently, Kaiser and coworkers reported enantiomeric specificity in the reaction of cyclohexaamylose with 3-carboxy-2,2,5,5-tetramethyl-pyrrolidin-l-oxy m-nitrophenyl ester (1), a spin label useful for identifying enzyme-substrate interactions (Flohr et al., 1971). In this case, the catalytic mechanism is identical to the scheme derived for the reactions of the cycloamyloses with phenyl acetates. In fact, the covalent intermediate, an acyl-cyclohexaamylose, was isolated. Maximal rate constants for appearance of m-nitrophenol at pH 8.62 (fc2), rate constants for hydrolysis of the covalent intermediate (fc3), and substrate binding constants (Kd) for the two enantiomers are presented in Table VIII. Significantly, specificity appears in the rates of acylation (fc2) rather than in either the strength of binding or the rate of deacylation. 

Yudushkin, I. A., Schleifenbaum, A., Kinkhabwala, A., Neel, B. G., Schultz, C. and Bastiaens, P. I. H. (2007). Live-cell Imaging of enzyme-substrate interaction reveals spatial regulation of PTP1B. Science 315, 115-119. 

R. Wolfenden, Conformational Aspects of Inhibitor Design Enzyme-Substrate Interactions in the Transition State , Bioorg Med. Chem. 1999, 7, 647-652... 

One approach, which has been used extensively in pharmacological areas [27], is to look for characteristic patterns of positive and negative regions that may enhance or inhibit a certain type of activity. The early stages of chug-receptor and enzyme-substrate interactions, in which the participants recognize each other through their outer electrostatic potentials, can be analyzed in this manner [28]. We were able, on this basis, to find qualitative trends in the toxicities of chlorinated dibenzo-p-dioxins and related systems [29]. 

Mitsiou and Stunnenberg, 2003). Moreover, enzyme-substrate interactions could be disrupted by missense mutations that are located throughout the protein. A clear example of this are the mutations in SRCl, which affect its interaction with CBP, but are located outside the CID region of SRCl (Rowan et al 2000 Wu et al., 2004) (see also above and Fig. lb). 

It is a common understanding that the spatial arrangements of the substituents of a molecule have an crucial effect on whether an enzyme can accept the compound as a substrate. The effect of configuration on the difference of reactivities of enantiomers may be evaluated, as the two enantiomers can be separated and treated as individual starting materials and their products. In fact, promising models of enzyme-substrate interactions have been proposed that permit successful interpretation of the difference of reactivities between a given pair of enantiomers [29,30]. On the other hand, analysis of the reactivity of the conformational isomers of a substrate is rather difficult,because conformers are readily interconvertible under ordinary enzymatic reaction conditions. 

Also a new family of inhibitors exemplified by SUAM 1221 65 [75] has been described in which a pyrrolidinylcarbonyl function at the PI site is the crucial entity for enzyme recognition, giving rise to the transition state analog of the enzyme-substrate interaction. 

We will also begin to explore other biological interactions such as ligand receptor binding, DNA hybridization, enzyme substrate interactions, or protein DNA complexation to further evaluate the generality of our approach. (From Lyon, 2000). 

Szklarz, G. D. and Paulsen, M. D. (2002) Molecular modeling of cytochrome P450 1A1 enzyme-substrate interactions and substrate binding affinities.. /. Biomol. Struct. Dyn. 20, 155-162. 

M. N. Namchuk and S. G. Withers, Mechanism of Agrobacterium p-glucosidase Kinetic studies of the role of noncovalent enzyme/substrate interactions, Biochemistry, 34 (1995) 16194-16202. 

With the support of quantum mechanics this proteolysis study has readily shown that fluorinated amino acid side chains are able to direct enzyme substrate interactions, which can have an influence on proteolytic stability. Depending on the absolute stereochemistry and on the position within the sequence, aTfm amino acids can considerably stabilize peptides against proteolysis. The unique electrostatic properties of carbon-bound fluorine, however, may also induce a contrary effect. The conformational restrictions of C -dialkylation seem to be partly dimin-ishable by the electrostatic consequences of fluorination. With this knowledge. 

---