Enzyme activation substrate complex

In this model, a molecule of enzyme (E) can bind one molecule of substrate (S) and/ or one molecule of activator (A). Equilibrium constants for the dissociation reactions ES <-> E + S, EAS EA + S, EA E + A and EAS <-> ES + A, are K , Kms, Ka and Kma respectively. The rate constants k and k are the rate constants for reactions ES —> E + P (product) and EAS —> EA + P respectively. The reaction scheme is based on the assumption that equilibrium between enzyme, substrate and activator, and their complexes is set up almost immediately and during the time required to measure initial velocity. Also, the higher concentrations of S and A than total enzyme concentration, as well as the velocities of product formation from the enzyme-substrate and enzyme-activator-substrate complexes as a velocity limiting steps in transformation S — P, were assumed. The rate constants k and k are related to the parameters Vi and V2 through following equations ... 

The results obtained from Lineweaver-Burk plots, are used for calculation of kinetic constants. The secondary plots of the slopes and intersects vs. activator concentrations are not linear (data not shown), but the reciprocal of the change in slope and intercept (Aslope and Ainiercept) that are determined by subtracting the values in the paesence of activator from that in its absence, are hnear. The intercepts of a plot 1/Aslope and 1/Airtercept 1/ [Al ] on 1/A axis, and intercepts of both plots on l/[ Al ] axis are used for calculating equilibrium constants Kms and Kma for dissociation of formed binary enzyme-activator (Al ) and ternary enzyme activator- substrate complexes (Figure 4). The calculated values for constants are (0.904 + 0.083) mM and (8.56 + 0.51) mM, respiectively. 

The inhibition process in general may be represented by the following six-step scheme (a similar scheme may be used for activation-see problem 10-12), in which I is the inhibitor, El is a binary enzyme-inhibitor complex, and EIS is a ternary enzyme-inhibitor-substrate complex. 

Although the mechanisms of enzyme activity are complex and not fully understood in most cases, a simple theory called the lock-and-key model (see Figure 21.8) seems to fit many enzymes. This model postulates that the shapes of the reacting molecule (the substrate) and the enzyme are such that they fit together much as a key fits a specific lock. The substrate and enzyme attach to each other in such a way that the part of the substrate where the reaction is to occur occupies the active site of the enzyme. After the reaction occurs, the products are liberated and the enzyme is ready for a new substrate. We can represent enzyme catalysis by the following steps ... 

Protein phosphatase 2A phosphatase activator, also known as Phosphotyrosyl phosphatase activator, PTPA, a conserved protein from yeast to humans. Its enzymatic activity as a peptidyl prolyl cis/trans isomerase has recently been identified, making it the fourth family of the enzyme class EC 5.2.1.8. Its isomerase activity can be stimulated by Mg /ATP. The three-dimensional structure of PTPA does not resemble those of other families of peptidyl prolyl cis/trans isomerases as an aU-helical fold dominates the two-domain organization of the enzyme. In the enzyme-peptide substrate complex, the peptide binds at the interface of a peptide-induced PTPA dimer. Apparently, protein phosphatase 2A activation and peptidyl prolyl cis/trans isomerase activity of PTPA are functionally linked in vitro [N. LeuUiot et al., J. Mol. Cell 2006, 23, 413]. 

Enzyme and substrate first reversibly combine to give an enzyme-substrate (ES) complex. Chemical processes then occur in a second step with a rate constant called kcat, or the turnover number, which is the maximum number of substrate molecules converted to product per active site of the enzyme per unit time. The kcat is, therefore, a rate constant that refers to the properties and reactions of the ES complex. For simple reactions kcat is the rate constant for the chemical conversion of the ES complex to free enzyme and products. 

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. 

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. 

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... 

Fibrinolytics. Figure 3 Plasminogen activation (a) Kinetics of plasminogen activation by uPA (urokinase-type) and tPA (tissue-type) plasminogen activators. Effect of fibrin (b) Ternary complex formation between enzyme (tPA), substrate (Pg) and cofactor (F) Abbreviations plasmin (P), fibrin (F), plasminogen (Pg). Plasmin, formed in time, is expressed in arbitrary units. 

Since the pioneering work of Kleymann et al. (2002), Betz et al. (2002), Baumeister et al. (2007), and Crute et al. (2002), who showed that compounds identified as inhibitors of the helicase-primase enzyme complex could alleviate herpesvirus-induced disease in animal models, the attention of researchers developing antiviral compounds has been drawn more and more towards the virus-encoded helicases, particularly those of Herpes viruses and of RNA viruses such as Hepatitis C Virus (HCV) and SAKS coronavirus (SARS-CoV). Enzyme activity is usually assayed by measuring NTPase activity in the presence of an appropriate nucleic acid co-substrate although, more recently, novel fiuorimetric and luminescence principles have been applied to the measurement of strand unwinding and/or translocation of the protein along the nucleic acid (Frick 2003, 2006). 

To clarify the characteristics of AMDase, the effects of some additives were examined using phenylmalonic acid as the representative substrate. The addihon of ATP and coenzyme A did not enhance the rate of the reaction, different from the case of malonyl-CoA decarboxylase and others in those, ATP and substrate acid form a mixed anhydride, which in turn reacts with coenzyme A to form a thiol ester of the substrate. In the present case, as both ATP and CoA-SH had no effect, the mechanism of the reaction will be totally different from the ordinary one described above. It is well estabhshed that avidin is a potent inhibitor of the formation of the biotin-enzyme complex. In the case of AMDase, addition of avidin has no influence on the enzyme activity, indicating that AMDase is not a biotin enzyme. 

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