262 Enzymatic properties of LYS158, LLE159 and ILE160 mutants of single-chain urokinase-type plasminogen activator

114

262 ENZYMATIC

AND ILE=' PROPERTIES OF LYS 158, ILE15' MUTANTS OF SINGLE-CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR H.R. Lijnen, B. Van Hoef, E. Demarsin L. Nelles, and D. Collen Center for Thrombosis and Vascular Research, University of Leuven, Belgium. Single-chain

urokinase-type

plasminogen

activator

(scu-PA) is converted to urokinase by cleavage of 158-Ile159 the Lys peptide bond with plasmin. resistant Lys 158 mutants (Gl~l~~ or Cleavage reduced catalytic GUY158) have a lo- to 20-fold efficiency

(k2/$)

al.,

J. Biol.

1987,

scu-PA-Arg similar

158 to

towards

plasminogen

Chem.,

262,

->

Arg

(LYs158 wild-type

(Nelles

et

5682).

mutant) scu-PA-Lys 158 :

activity IU/mg; 0.21

fibrin

plates

: 48,000

efficiency towards -1 -1 . scu-PA-Pro15' mM s

vs 0.64 159 are inactive

GUY 0.002

mM-'s-')

towards and

are

vs

60,000

plasminogen

:

and scu-PA-

plasminogen converted

(k2/s to

<

inactive

two-chain derivatives by plasmin. scu-PA158 16' (with Lye.158 -> Gly and Ile16' -> rLYS GUY Lys mutation) has kinetic properties comparable to scu-PA-Gly 158 . These of Arg) are

is very specific

on catalytic

results

a positive but

also

essential

convertibility

suggest side the for

that chain

presence the

to active

not

only

in position of

Ile

activity two-chain

the

presence

158 (Lys in position

of scu-PA urokinase.

or 159

and its