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.
Liu
J N
JN
Vascular Research Laboratory, Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA. liu@mbcrr.harvard.edu
Tang
W
W
Sun
Z Y
ZY
Kung
W
W
Pannell
R
R
Sarmientos
P
P
Gurewich
V
V
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