A site-directed mutagenesis of pro-urokinase which substantially reduces its intrinsic activity.
Single-chain urokinase-type plasminogen activator or pro-urokinase is a zymogen with an intrinsic catalytic activity which is greater than that of most other zymogens. To study the structural basis for this activity, a three-dimensional homology model was calculated using the crystallographic structure of chymotrypsinogen, and the structure-function relationship was studied using site-directed mutagenesis and kinetic analysis. This model revealed a unique Lys300 in pro-urokinase which could form a weak interaction with Asp355, adjacent to the active site Ser356. It was postulated that this lysine, by its epsilon-amino group, may serve to pull Ser356 close to the active position, thereby inducing the higher intrinsic activity of pro-urokinase. This was consistent with the published finding that a homologous lysine (Lys416) in single chain tissue plasminogen activator when mutated to serine induced some reduction in activity. To test this hypothesis, a site-directed mutant with a neutral residue (Lys300-->Ala) was produced and characterized. The Ala300-pro-urokinase had a 40-fold lower amidolytic activity than that of pro-urokinase. It was also stable in plasma at much higher concentrations than pro-urokinase, reflecting much attenuated plasminogen activation. Plasmin activatability was comparable to that of pro-urokinase, but the resultant two-chain derivative (Ala300-urokinase) had a lower enzymatic activity (approximately 33% that of urokinase) due to a reduction of kcat. Interestingly, the KM of two-chain Ala300-urokinase against plasminogen was 5.8-fold lower than that of urokinase, being similar to that of pro-urokinase which has a KM about 5-fold lower than urokinase. In conclusion, the hypothesis that Lys300 is a key structural determinant of the high intrinsic activity of pro-urokinase was confirmed by these studies. This residue also appears to be important for the full expression of the enzymatic activity of urokinase.
Vascular Research Laboratory, Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA. liu@mbcrr.harvard.edu
J N
JN
Liu
W
W
Tang
Z Y
ZY
Sun
W
W
Kung
R
R
Pannell
P
P
Sarmientos
V
V
Gurewich
D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry*; D005342:Fibrinolysis; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D014568:Urokinase-Type Plasminogen Activator