Hydrolysis kinetics substrates

In our previous work [63], we studied the hydrolysis kinetics of lipase from Mucor javanicus in a modified Lewis cell (Fig. 4). Initial hydrolysis reaction rates (uri) were measured in the presence of lipase in the aqueous phase (borate buffer). Initial substrate (trilinolein) concentration (TLj) in the organic phase (octane) was between 0.05 and 8 mM. The presence of the interface with octane enhances hydrolysis [37]. Lineweaver-Burk plots of the kinetics curve (1/Uj.] = f( /TL)) gave straight lines, demonstrating that the hydrolysis reaction shows the expected kinetic behavior (Michaelis-Menten). Excess substrate results in reaction inhibition. Apparent parameters of the Michaelis equation were determined from the curve l/urj = f /TL) and substrate inhibition was determined from the curve 1/Uj.] =f(TL) ... 

Because the preceding chromogenic assay rely on choline quantitation, the hydrolysis of substrates with headgroups other than choline cannot be followed. To circumvent this problem, another useful protocol was devised whereby the phosphorylated headgroup produced by the PLCBc hydrolysis is treated with APase, and the inorganic phosphate (Pi) that is thus generated is quantitated by the formation of a blue complex with ammonium molybdate/ascorbic acid 5 nmol of phosphate may be easily detected. This assay, which may also be performed in a 96-well format, has been utilized to determine the kinetic parameters for the hydrolysis of a number of substrates by PLCBc [37,38]. 

Activation of a-D-mannosidase from the limpet by Zn2+ and Cl-provides a particularly good example of the ways in which the kinetics of hydrolysis may be altered. Fig. 2 shows the effect of Zn2+, Cl-, or both, on the velocity of hydrolysis of substrate at varying concentration. Inspection of die curves reveals that Zn2+ increases the affinity of the enzyme for the substrate (competitive type of effect), whereas the main effect of Cl- is to increase the rate of hydrolysis (non-competitive effect). 

The problem of modeling the hydrolysis kinetics is complicated by the fact that cellulose is a solid substrate consequently, the reaction can be surface limited (5,12). Furthermore, some sites are more susceptible to hydrolysis—e.g., the amorphous regions as well as specific regions of the crystalline cellulose such as edges, corners, and dislocations. Several investigators (17,20,36) have suggested that the kinetic model should be based on a shrinking site model in which the number of susceptible... 

Further progress in understanding this effect, as well as others, of the physical structure of cellulose on enzymatic degradation may be expected from combining physicochemical and morphological techniques and from kinetic measurements in heterogeneous enzymatic hydrolysis, applying substrates of well-defined physical structure and isolated components of the enzyme systems. 

Kinetics of Model Substrates. In an effort to better understand enzymatic kinetics within reversed micelles, a-chymotrypsin hydrolysis of a model substrate, GPANA, was studied in CTAB reversed micelles. The hydrolysis kinetics of substrates by a-chymotrypsin is described by the Michaells-Menten formulation... 

Figure 12.5-18. Schematic representation of the new extended kinetic model of peptidase-catalyzed hydrolysis of substrate mimetics according to Thormann et o/.[19S. ...

The kinetic probes 1 and 2 were selected because the hydrolysis of substrates is characterized by pH-independent rates between pH = 2-6. This is also the pH region in which at-PMAA undergoes a compact-coil to random-coil transition. By changing the hydrophobicity of substrates through variation of R, / i and R2 in I and 2, it is also possible to elucidate the role of hydrophobic microdomains inside PMAA. 

Summary rac-l-(4-Fluorophenyl)-l-methyl-l-sila-2-cyclohexanone (rac-1) and rac-(SiS, C/ /Sii ,CS)-2-acetoxy-l-(4-fluorophenyl)-l-methyl-l-silacycIohexane [rac-(Si5,Ci /Siiil,C5)-3a] were synthesized as substrates for stereoselective microbial transformations. Resting free cells of the yeast Saccharomyces cerevisiae (DSM 11285) and growing cells of the yeasts Trigonopsis variabilis (DSM 70714) and Kloeckera corticis (ATCC 20109) were found to reduce rac- diastereoselectively to yield mixtures of the enantiomers (S S,CR)- and (SLR,C5)-l-(4-fluorophenyl)-l-methyl-1-sila-2-cyclohexanol [(Si5,C/ )-2a and (SLR,CS)-2a]. In the case of Kloeckera corticis (ATCC 20109), diastereoselective reduction of rac-1 gave a quasi-racemic mixture of (Si5,CR)-2a and (SiR,CS)-2a (diastereomeric purity 95 % de, yield 95 %). Enantioselective ester hydrolysis (kinetic resolution) of the 2-acetoxy-l-silacyclohexane rac-(Si5,CR/Si/ ,C5)-3a yielded the optically active l-sila-2-cyclohexanol (Si/ ,C5)-2a [enantiomeric purity >99% ee yield 56 % (relative to (Si, C5)-3a)]. 

Asteriou, T., Gouley, F., Deschrevel, B., and Vincent, J. C. (2002). In Influence of Substrate and Enzyme Concentrations on Hyaluronan Hydrolysis Kinetics Catalyzed by Hyaluronidase. J. F. Hyaluronan, G. O. Kennedy, and P. A. Phillips Williams (Eds.). Woodhead Publishing, Wrexham, Wales, vol. 1, pp. 249-252. 

Asteriou, T., Deschrevel, B., Gouley F., Vincent, J.C. (2002) Influence of substrate and enzyme concentration on hyaluronan hydrolysis kinetics catalyzed by hyaluronidase, in Hyaluronan Proceedings of an International Meeting, September 2000, North East Wales Institute, UK (eds J.F. Kennedy, G.O. Phillips, P.A. Williams, V.C. Hascall), Woodhead Publishing Ltd, Cambridge, pp. 249-252. 

Additionally, global fitting of this equation to the kinetic data was able to confirm that the inhibitor imidazole had an irreversible component to its inhibition of P-galactosidase (Kim et al., 2003) as the simple insertion of modifier terms into equation 35 was unable to describe the effect of the inhibitor on the enzyme. While the hydrolysis of substrate tended towards zero in the absence of imidazole, the introduction of the inhibitor stopped the enzymatic activity in a concentration dependent manner. Therefore it was reasoned that a certain fraction of the inhibitor bound enzyme population was inactivated by this process (Equation 37). 

In this case study, an enzymatic hydrolysis reaction, the racemic ibuprofen ester, i.e. (R)-and (S)-ibuprofen esters in equimolar mixture, undergoes a kinetic resolution in a biphasic enzymatic membrane reactor (EMR). In kinetic resolution, the two enantiomers react at different rates lipase originated from Candida rugosa shows a greater stereopreference towards the (S)-enantiomer. The membrane module consisted of multiple bundles of polymeric hydrophilic hollow fibre. The membrane separated the two immiscible phases, i.e. organic in the shell side and aqueous in the lumen. Racemic substrate in the organic phase reacted with immobilised enzyme on the membrane where the hydrolysis reaction took place, and the product (S)-ibuprofen acid was extracted into the aqueous phase. 

Substrate and product inhibitions analyses involved considerations of competitive, uncompetitive, non-competitive and mixed inhibition models. The kinetic studies of the enantiomeric hydrolysis reaction in the membrane reactor included inhibition effects by substrate (ibuprofen ester) and product (2-ethoxyethanol) while varying substrate concentration (5-50 mmol-I ). The initial reaction rate obtained from experimental data was used in the primary (Hanes-Woolf plot) and secondary plots (1/Vmax versus inhibitor concentration), which gave estimates of substrate inhibition (K[s) and product inhibition constants (A jp). The inhibitor constant (K[s or K[v) is a measure of enzyme-inhibitor affinity. It is the dissociation constant of the enzyme-inhibitor complex. 

Esterases have a catalytic function and mechanism similar to those of lipases, but some structural aspects and the nature of substrates differ [4]. One can expect that the lessons learned from the directed evolution of lipases also apply to esterases. However, few efforts have been made in the directed evolution of enantioselective esterases, although previous work by Arnold had shown that the activity of esterases as catalysts in the hydrolysis of achiral esters can be enhanced [49]. An example regarding enantioselectivity involves the hydrolytic kinetic resolution of racemic esters catalyzed by Pseudomonasfluorescens esterase (PFE) [50]. Using a mutator strain and by screening very small libraries, low improvement in enantioselectivity was... 

In an asymmetric synthesis, the enantiomeric composition of the product remains constant as the reaction proceeds. In practice, ho vever, many enzymatic desymmetrizations undergo a subsequent kinetic resolution as illustrated in Figure 6.5. For instance, hydrolysis of a prochiral diacetate first gives the chiral monoalcohol monoester, but this product is also a substrate for the hydrolase, resulting in the production of... 

The main application of the enzymatic hydrolysis of the amide bond is the en-antioselective synthesis of amino acids [4,97]. Acylases (EC 3.5.1.n) catalyze the hydrolysis of the N-acyl groups of a broad range of amino acid derivatives. They accept several acyl groups (acetyl, chloroacetyl, formyl, and carbamoyl) but they require a free a-carboxyl group. In general, acylases are selective for i-amino acids, but d-selective acylase have been reported. The kinetic resolution of amino acids by acylase-catalyzed hydrolysis is a well-established process [4]. The in situ racemization of the substrate in the presence of a racemase converts the process into a DKR. Alternatively, the remaining enantiomer of the N-acyl amino acid can be isolated and racemized via the formation of an oxazolone, as shown in Figure 6.34. 

Stereoinversion Stereoinversion can be achieved either using a chemoenzymatic approach or a purely biocatalytic method. As an example of the former case, deracemization of secondary alcohols via enzymatic hydrolysis of their acetates may be mentioned. Thus, after the first step, kinetic resolution of a racemate, the enantiomeric alcohol resulting from hydrolysis of the fast reacting enantiomer of the substrate is chemically transformed into an activated ester, for example, by mesylation. The mixture of both esters is then subjected to basic hydrolysis. Each hydrolysis proceeds with different stereochemistry - the acetate is hydrolyzed with retention of configuration due to the attack of the hydroxy anion on the carbonyl carbon, and the mesylate - with inversion as a result of the attack of the hydroxy anion on the stereogenic carbon atom. As a result, a single enantiomer of the secondary alcohol is obtained (Scheme 5.12) [8, 50a]. 

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