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. 2011 Feb 3;20(4):670–683. doi: 10.1002/pro.596

Table II.

Kinetic Constants

Inline graphic
Construct KM (μM) kcat (min−1) kcat/KM (μM−1 min−1)
wild-type 1-303 162 68 0.42
Lid-domain mutants
 1-303 Leu169Ser, Leu176Ser 136 48 0.35
 9-393 Leu169Ser, Leu176Ser 88 27 0.31
 9-297 Leu169Ser, Leu176Ser 126 26 0.21
 1-303 Leu171Gln 105 51 0.48
 1-303 Leu167Gln, Leu171Gln 84 59 0.71
 1-303 Leu167Gln, Leu174Gln 84 70 0.83
 1-303 Leu171Gln, Leu174Gln 89 47 0.52
Lid-domain + Surface mutants
 1-303 Leu169Ser, Leu176Ser, Lys36Ala 124 51 0.41
 1-303 Leu169Ser, Leu176Ser, Lys160Ala 90 30 0.33
 1-303 Leu169Ser, Leu176Ser, Lys165Ala 137 27 0.2
 1-303 Leu169Ser, Leu176Ser, Lys226Ala 110 38 0.35
 1-303 Leu169Ser, Leu176Ser, Lys36Ala, Lys226Ala 123 30 0.25
Inline graphic
wild-type 1-303 * * 0.09
mutant 1-303, Leu169Ser, Leu176Ser * * 0.1

Kinetic constants of the various MGL constructs using 4-methylumbelliferyl butyrate or umbelliferyl arachidonate as substrates. Values for the 4-methylumbelliferyl butyrate substrate are the average of 2 or 4 separate assays. kcat/KM values for the umbelliferyl arachidonate substrate are the average values for the hydrolysis of five different substrate concentrations at [S] < KM.

(*)The solubility limit of the umbelliferyl arachidonate substrate did not allow for the determination of KM and kcat.