Question

we did the experiment To determine the effect of altering the ester substrate on the rate of the esterase hydrolysis reaction, using three different esters: ethyl acetate, ethyl propanoate, and ethyl butanoate. The rate of the reaction will be followed by measuring the amount of alkali (KOH) required to neutralise the acid liberated during hydrolysis.

1.Ethyl benzoate,unlike the substrates you have used in the practical, is derived from benzoic acid, and has a ring structure.This ester is not lysed by the esterase you have used. provide a reason why this happens.

2.What can you deduce about the shape of the active site of the enzyme based on your results and the data on ethyl benzoate.

fx 20*M24 Table 1: KOH (conc. 20 mM) required to neutralise the acid produced upon esterase-catalysed hydrolysis of different ester substrates KOH used 1. 50 ml phosphate buffer; 2. 2.5 ml Ethyl butanoate 1. 50 ml phosphate buffer 2. 2.5 ml Ethyl propanoate 1.50 ml phosphate buffer; 2. 2.5 ml Ethyl acetate 3. 2 ml of esterase 3. 2 ml of esterase. 3.2 ml of esterase Cumulative Total Cumulative Total Drops Cumulative Total Drops Drops Vol mmol (ml) (necv) Vol mmol per Vol mmol per ml) (nsv)20sec DropsV ml) (nscv) 20sec Drops 20sec 0 0.189 3.783 5 10 0.378 7.567 0.190 3.800 0.114 2.270 0.189 3.783 0.570 11.400 10 0.912 18.240 0.265 5.297 18 0.681 13.620 9 0.303 6.053 0.984 19.673 30 1.14022.800 10 0.378 7.567 0.454 9.080 1.173 23.457 .44428.880 1.400 27.997 1.710 34.200 2.090 41.800 2.39447.880 2.66053.200 2.888 57.760 3.11662.320 0.530 10.593 1589 31.780 10 1.778 35.563 1.967 39.347 42 15 0.568 11.350 0.643 12.863 18 0.681 13.620 63 52 70 2.081 41.617 0.757 15.133 2.194 43.887 6 0.870 17.403 2.346 46.913 90 0.908 18.160 0.984 19.673 3.42068.400 67 2.53550.697 7 3.68673.720 104 3.95279.040 70 2.648 52.967 Finished? Proceed to B. Km and Vmax (calculation) Start here effect on rato

Answer 1

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