Enzyme-activator complexes

According to Supuran and co-workers (199,204), an enzyme-activator complex may be formed in which the activator facilitates the proton transfer. A general reaction scheme is presented in Scheme 9. 

When nuclease-(1-126) is mixed with nuclease-T-(49,50-149) (the mixture of nuclease-T-(49-149), and nuclease-T-(50-149) ], enzymic activity is generated at the level of nuclease-T and the formation of ordered structure is also indicated (80). After incubation of the enzymically active mixture in the presence of pdTp and Ca2+ with trypsin for a fairly long time, approximately 50% of the original enzymic activity remains. The enzymically active complex thus formed is the same as nuclease-T and is composed of the two fragments nuclease-T-(6-48) and nuclease-T-(49-149) [or nuclease-T-(50-149)] (80). Therefore, nuclease-T-(49, 50-149) binds to the nuclease-T-(6-48) portion of nuclease-(1-126) forming an enzymically active structure like nuclease-T, and the redundant residues 49-126 of nuclease-(1-126) extend in a disordered manner from the ordered structure. 

Mammalian alpha-amylases are activated by monovalent anions, especially chloride. Equations, similar to those for enzyme inhibition (see p. 290), may be written for the formation of enzyme—activator complexes, and kinetic parameters may be derived. Dissociation constants of salivary alpha-amylase—anion complexes have been determined. ... 

Canavalia ensiformis, Phaseolus vulgaris, and Triticum vulgare react with arylamidase, alkaline phosphatase, y-glutamyltransferase, and cholinesterase of human sera by formation of enzymically active complexes. Arylamidase, alkaline phosphatase, and cholinesterase react more intensely in sera of patients with liver and neoplastic diseases. 

Simple geometrical considerations illustrated in Figure 5 show that kinetic data could be used for graphical determination of the activator concentration that gives a reaction rate equal to the half of saturation concentration of activator (A50), as well as the dissociation constant Ka for enzyme-activator complex. If the abscissa variable is 1/[A], then the intercept is - 1/ [A50], where [A50] is activator concentration that gives a rate equal to the half that at a saturating concentration of activator. 

All organisms seem to have an absolute need for magnesium. In plants, the magnesium complex chlorophyll is the prime agent in photosynthesis. In animals, magnesium functions as an enzyme activator the enzyme which catalyses the ATP hydrolysis mentioned above is an important example. 

The reaction is proposed to proceed from the anion (9) of A/-aminocatbonylaspattic acid [923-37-5] to dehydrooranate (11) via the tetrahedral activated complex (10), which is a highly charged, unstable sp carbon species. In order to design a stable transition-state analogue, the carboxylic acid in dihydrooronate (hexahydro-2,6-dioxo-4-pyrimidinecarboxylic acid) [6202-10-4] was substituted with boronic acid the result is a competitive inhibitor of dibydroorotase witb a iC value of 5 ]lM. Its inhibitory function is supposedly due to tbe formation of tbe charged, but stable, tetrabedral transition-state intermediate (8) at tbe active site of tbe enzyme. 

This class of inhibitors usually acts irreversibly by permanently blocking the active site of an enzyme upon covalent bond formation with an amino acid residue. Very tight-binding, noncovalent inhibitors often also act in an irreversible fashion with half-Hves of the enzyme-inhibitor complex on the order of days or weeks. At these limits, distinction between covalent and noncovalent becomes functionally irrelevant. The mode of inactivation of this class of inhibitors can be divided into two phases the inhibitors first bind to the enzyme in a noncovalent fashion, and then undergo subsequent covalent bond formation. 

Usually, a rapid binding step of the inhibitor I to the enzyme E leads to the formation of the initial noncovalent enzyme-inhibitor complex E-I. This is usually followed by a rate determining catalytic step, leading to the formation of a highly reactive species [E—I ]. This species can either undergo reaction with an active site amino acid residue of the enzyme to form the covalent enzyme-inhibitor adduct E—I", or be released into the medium to form product P and free active enzyme E. 

The often fast binding step of the inhibitor I to the enzyme E, forming the enzyme inhibitor complex E-I, is followed by a rate-determining inactivation step to form a covalent bond. The evaluation of affinity labels is based on the fulfillment of the following criteria (/) irreversible, active site-directed inactivation of the enzyme upon the formation of a stable covalent linkage with the activated form of the inhibitor, (2) time- and concentration-dependent inactivation showing saturation kinetics, and (3) a binding stoichiometry of 1 1 of inhibitor to the enzyme s active site (34). 

A significant difference between pseudoirreversible inhibitors and mechanism-based inactivators is the reversibiUty of the inactivation. A complete evaluation of the mechanism involved would require evidence not only for the covalent enzyme-inhibitor complex, but also for its decomposition products and its rate of reactivation. It is often difficult to identify the active site amino acid residue covalently linked to the inhibitor because of the instabiUty of the complex. 

Inhibitors as well as substrates bind in this crevice between the domains. From the numerous studies of different inhibitors bound to serine pro-teinases we have chosen as an illustration the binding of a small peptide inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH to a bacterial chymotrypsin (Figure 11.9). The enzyme-peptide complex was formed by adding a large excess of the substrate Ac-Pro-Ala-Pro-Tyr-CO-NHz to crystals of the enzyme. The enzyme molecules within the crystals catalyze cleavage of the terminal amide group to produce the products Ac-Pro-Ala-Pro-Tyr-COOH and NHs. The ammonium ions diffuse away, but the peptide product remains bound as an inhibitor to the active site of the enzyme. 

The Protein Data Bank, PDB ID 1A71. Colby, T. D., Bahnson, B. J., Chin, J. K., Klinman, J. P, Goldstein, B. M., Active Site Modifications in a Double Mutant of Liver Alcohol Dehydrogenase Structural Studies of Two Enzyme-Ligand Complexes. To be published. 

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

The catalytically active enzyme substrate complex is an interactive structure in which the enzyme causes the substrate to adopt a form that mimics the transition-state intermediate of the reaction. Thus, a poor substrate would be one that was less effective in directing the formation of an optimally active enzyme transition-state intermediate conformation. This active conformation of the enzyme molecule is thought to be relatively unstable in the absence of substrate, and free enzyme thus reverts to a conformationally different state. 

Glycogen synthase also exists in two distinct forms which can be interconverted by the action of specific enzymes active, dephosphorylated glycogen synthase I (glucose-6-P-independent) and less active phosphorylated glycogen synthase D (glucose-6-P-dependent). The nature of phosphorylation is more complex with glycogen synthase. As many as nine serine residues on the enzyme appear to be subject to phosphorylation, each site s phosphorylation having some effect on enzyme activity. 

It seems reasonable that an enzyme which used poraaminobenzoic acid as a substrate might be deceived by sulfanilamide. The two compounds are very similar in size and shape and in many chemical properties. To explain the success of sulfanilamide, it is proposed that the amide can form an enzyme-substrate complex that uses up the active centers normally occupied by the natural substrate. 

The stability of the enzyme-polymer complex and its dissociation upon the variation of pH depends on the structural and other physico-chemical properties of CP and enzyme molecule. Thus, a Biocarb-T heteroreticular biosorbent (Fig. 26) is characterized by a stability of its complex with ot-amylase (under the condition of its stabilization) in acid solutions and a complete dissociation of the complex during isolation of the active enzyme at pH 7-8. 

Acyloins (a-hydroxy ketones) are formed enzymatically by a mechanism similar to the classical benzoin condensation. The enzymes that can catalyze reactions of this type arc thiamine dependent. In this sense, the cofactor thiamine pyrophosphate may be regarded as a natural- equivalent of the cyanide catalyst needed for the umpolung step in benzoin condensations. Thus, a suitable carbonyl compound (a -synthon) reacts with thiamine pyrophosphate to form an enzyme-substrate complex that subsequently cleaves to the corresponding a-carbanion (d1-synthon). The latter adds to a carbonyl group resulting in an a-hydroxy ketone after elimination of thiamine pyrophosphate. Stereoselectivity of the addition step (i.e., addition to the Stand Re-face of the carbonyl group, respectively) is achieved by adjustment of a preferred active center conformation. A detailed discussion of the mechanisms involved in thiamine-dependent enzymes, as well as a comparison of the structural similarities, is found in references 1 -4. 

In general, pyruvate decarboxylase (EC 4.1.1.1) catalyzes the decarboxylation of a 2-oxocar-boxylic acid to give the corresponding aldehyde6. Using pyruvic acid, the intermediately formed enzyme-substrate complex can add an acetyl unit to acetaldehyde already present in the reaction mixture, to give optically active acetoin (l-hydroxy-2-butanone)4 26. Although the formation of... 

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