Biocatalysis steady state kinetics

Biocatalyst inhibitors I are substrate-like molecules that interact with a given biocatalyst and interfere with the progress of biocatalysis. Inhibitors usually act in one of three ways, either by competitive inhibition, non-competitive inhibition or uncompetitive inhibition. The mode of inhibition is different in each case and as a result a different steady state kinetic scheme is required to account for each mode of inhibition. Consequently, each mode of inhibition is characterised by a different steady state kinetic equation that gives rise to a different graphical output of V versus [S] data, as we will show below. These substantial differences in graphical output can be used to diagnose the type of inhibition if unknown. 

The Michaelis-Menten equation (8.8) and the irreversible Uni Uni kinetic scheme (Scheme 8.1) are only really applicable to an irreversible biocatalytic process involving a single substrate interacting with a biocatalyst that comprises a single catalytic site. Hence with reference to the biocatalyst examples given in Section 8.1, Equation (8.8), the Uni Uni kinetic scheme is only really directly applicable to the steady state kinetic analysis of TIM biocatalysis (Figure 8.1, Table 8.1). Furthermore, even this statement is only valid with the proviso that all biocatalytic initial rate values are determined in the absence of product. Similarly, the Uni Uni kinetic schemes for competitive, uncompetitive and non-competitive inhibition are only really applicable directly for the steady state kinetic analysis for the inhibition of TIM (Table 8.1). Therefore, why are Equation (8.8) and the irreversible Uni Uni kinetic scheme apparently used so widely for the steady state analysis of many different biocatalytic processes A main reason for this is that Equation (8.8) is simple to use and measured k t and Km parameters can be easily interpreted. There is only a necessity to adapt catalysis conditions such that... 

In this case. Equation (8.26) and the irreversible Uni Uni kinetic scheme for four catalytic sites is appropriate for the steady state kinetic analysis of homo-tetrameric, human mitochondrial MnSOD biocatalysis (Figure 8.8, Table 8.1) with one catalytic site containing one manganese ion per subunit. These sites are not only independent, but each turnover of a catalytic site involves one substrate superoxide radical being transformed to only one of two possible redox products depending upon the oxidation state of the manganese ion involved. 

So far in this chapter, the chemical biology reader has been introduced to examples of biocatalysts, kinetics assays, steady state kinetic analysis as a means to probe basic mechanisms and pre-steady-state kinetic analysis as a means to measure rates of on-catalyst events. In order to complete this survey of biocatalysis, we now need to consider those factors that make biocatalysis possible. In other words, how do biocatalysts achieve the catalytic rate enhancements that they do This is a simple question but in reality needs to be answered in many different ways according to the biocatalyst concerned. For certain, there are general principles that underpin the operation of all biocatalysts, but there again other principles are employed more selectively. Several classical theories of catalysis have been developed over time, which include the concepts of intramolecular catalysis, orbital steering , general acid-base catalysis, electrophilic catalysis and nucleophilic catalysis. Such classical theories are useful starting points in our quest to understand how biocatalysts are able to effect biocatalysis with such efficiency. 

The kinetic of the biocatalysis was determined before and after the immobilization of the enzyme on the nanofibrous membrane. The kinetic constants ( and Vniax) sured by studying the relationship between the steady-state current and the concentration... 

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