Functional evaluation of the structural features of proteases and their substrate in fibrin surface degradation
Citation
Kolev, K and Tenekedjiev, K and Komorowicz, E and Machovich, R, Functional evaluation of the structural features of proteases and their substrate in fibrin surface degradation, Journal of Biological Chemistry, 272, (21) pp. 13666-13675. ISSN 0021-9258 (1997) [Refereed Article]
A new model has been introduced to characterize the
action of a fluid phase enzyme on a solid phase substrate.
This approach is applied to evaluate the kinetics
of fibrin dissolution with several proteases. The model
predicts the rate constants for the formation and dissociation
of the protease-fibrin complex, the apparent order
of the association reaction between the enzyme and
the substrate, as well as a global catalytic constant (kcat)
for the dissolution process. These kinetic parameters
show a strong dependence on the nature of the applied
protease and on the structure of the polymerized substrate.
The kinetic data for trypsin, PMN-elastase, and
three plasminogen-derived proteases with identical catalytic
domain, but with a varied N-terminal structure,
are compared. The absence of kringle5 in des-kringle1–5-
plasmin (microplasmin) is related to a markedly lower
kcat (0.008 s21
) compared with plasmin and des-kringle1–4-
plasmin (miniplasmin) (0.039 s21
). The essentially identical
kinetic parameters for miniplasmin and plasmin
with the exception of kdiss, which is higher for miniplasmin
(81.8 s21 versus 57.6 s21
), suggest that the first four
kringle domains are needed to retain the enzyme in the
enzyme-fibrin complex. Trypsin, a protease of similar
primary specificity to plasmin, but with a different catalytic
domain, shows basically the same kcat as plasmin,
but its affinity to fibrin is markedly lower compared
with plasmin and even microplasmin. The latter suggests
that in addition to the kringle domains, the structure
of the catalytic domain in plasmin also contributes
to its specificity for fibrin. The thinner and extensively
branched fibers of fibrin are more efficiently dissolved
than the fibers with greater diameter and lower number
of branching points. When the polymer is stabilized
through covalent cross-linking, the kcat for plasmin and
miniplasmin is 2–4-fold higher than on non-cross-linked
fibrin, but the decrease in the association rate constant
for the formation of enzyme-substrate complex explains
the relative proteolytic resistance of the cross-linked
fibrin. Thus, the functional evaluation of the discrete
steps of the fibrinolytic process reveals new aspects of
the interactions between proteases and their polymer
substrate.