Purification and characterization of mutant miniPlasmin for thrombolytic therapy

Thrombosis Journal

Table 2 Kinetic parameters of wild-type and mutant mPlg*

mPlm	Active μM in 0.5 μM	Vmax (μM/Min)	Km (μM)	Kcat (Min^-1)	Kcat/Km	Kcat/Km Ratio
					(μM^-1Min^-1)
01_K698P	0.00375	11 ± 0.5	2145.3 ± 145.5	2933	1.37	0.29
02_WT	0.1265	207 ± 4.9	347 ± 25	1636	4.72	1
03_K698L	0.0685	126 ± 6.8	2459 ± 201	1839	0.75	0.16
04_K698F	0.124	97 ± 4.6	439 ± 59	782	1.78	0.38
05_K698D	0.046	17 ± 1	587 ± 83	370	0.63	0.13
06_K698W	0.0065	72 ± 8	9686 ± 1222	11077	1.14	0.24
07_K698T	0.007	24 ± 1	4107 ± 221	3429	0.83	0.18
08_K698N	0.1625	224 ± 4.4	811 ± 35	1378	1.7	0.36
09_K698I	0.399	152 ± 6.6	1587 ± 118	381	0.24	0.05
10_K698Q	0.129	213 ± 6.5	1406 ± 76	1651	1.17	0.25
11_K698E	0.1905	62 ± 2.8	2411 ± 162	325	0.13	0.03
12_K698V	0.154	202 ± 5.6	1609 ± 77	1312	0.82	0.17
13_K698G	0.108	11 ± 1.4	617 ± 181	102	0.17	0.03
14_K698M	0.127	221 ± 5.6	1664 ± 71	1740	1.05	0.22
15_K698Y	0.3545	33 ± 1.9	2451 ± 209	93	0.04	0.01
16_K698S	0.1405	39 ± 1.3	1776 ± 96	278	0.16	0.03
17_K698A	0.046	11 ± 0.7	885 ± 111	239	0.27	0.06

* Column 1 in Table 2 shows different mutants (WT: wild-type). Column 2 shows the active enzyme calculated from active site titration, which is an indication of either refolding efficiency or stability of the active zymogen. The concentration of the active enzyme was used to calculate Kcat. Columns 3-7 show the measured and calculated kinetic parameters; column 7 shows the ratio of the catalytic efficiency of different mutants compared with the wild-type, which is set to 1.

ISSN: 1477-9560