The role of carbohydrate side chains of plasminogen in its activation by staphylokinase

Айсина, Р.Б.; Мухаметова, Л.И.; Gershkovich, K.B.; Varfolomeyev, S.D.

doi:10.1016/j.bbagen.2005.07.007

Cited by 18 publications

(8 citation statements)

References 34 publications

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“…We applied the same methodology of conventional initial rate analysis to our kinetic data (Figure 2a,b,d,e; Supplementary Table S1) and compared the obtained values with those from the literature. 19,24,25,28,[34][35][36][37][38][39] Values similar to the reported range (Table 1) were obtained for both the turnover number kcat (0.011 ± 0.001 s -1 ) and Michaelis constant Km (21 ± 1 µM). Such results confirmed the consistency of our kinetic data with those previously reported.…”

Section: Biophysical Characterization Confirms Protein Qualitysupporting

confidence: 73%

“…We validated our kinetic data by first applying a conventional data analysis approach and obtained comparable values of underestimated steady-state kinetic parameters as those reported previously. 19,24,25,28,[34][35][36][37][38][39] It should be noted that some studies have reported values of kcat approximately 10-fold higher and out of the range of other consistent results. 22,23,26,27,43 However, those data were collected at elevated temperatures where exponentially higher rates are expected.…”

Section: Discussionmentioning

confidence: 99%

“…Although many prior reports have focused on the kinetic characterization of SAK and its modified variants, 19,[22][23][24][25][26][27][28][34][35][36][37][38][39]43 we were unable to find a single study reporting the analysis of SAK kinetics by a combination of steady-state and binding affinity datasets. Some studies conducted both types of experiments 19,23,26,40 but did not combine the data to obtain global estimates of kinetic parameters.…”

Section: Discussionmentioning

confidence: 99%

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Extended Mechanism and Rate-Limiting Step of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis

Toul

Nikitin

Marek

et al. 2021

Preprint

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The plasminogen activator staphylokinase is a fibrin-specific thrombolytic biomolecule and an attractive target for the development of effective myocardial infarction and stroke therapy. To engineer the protein rationally, a detailed understanding of the biochemical mechanism and limiting steps is essential. Conventional fitting to equations derived based on simplifying approximations may be inaccurate for complex mechanisms like that of staphylokinase. We employed a modern numerical approach of global kinetic data analysis whereby steady-state kinetics and binding affinity datasets were analyzed in parallel. Our approach provided an extended, revised understanding of the staphylokinase mechanism without simplifying approximations and determined the value of turnover number kcat of 117 s-1 that was 10,000-fold higher than that reported in the literature. The model further showed that the rate-limiting step of the catalytic cycle is the binding of staphylokinase to plasmin molecules, which occurs via an induced-fit mechanism. The overall staphylokinase effectivity is further influenced by the formation of an inactive staphylokinase.plasminogen complex. Here, we describe a quick and simplified guide for obtaining reliable estimates of key parameters whose determination is critical to fully understand the staphylokinase catalytic functionality and define rational strategies for its engineering. Our study provides an interesting example of how global numerical analysis of kinetic data can be used to better understand the mechanism and limiting factors of complex biochemical processes.

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Section: Biophysical Characterization Confirms Protein Qualitysupporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Extended Mechanism and Rate-Limiting Step of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis

Toul

Nikitin

Marek

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…We applied the same methodology of conventional initial rate analysis to our kinetic data (Figure a,b,d,e and Table S1) and compared the values obtained with those from the literature. ,,,,− Values similar to the reported range (Table ) were obtained for both the turnover number k cat (0.011 ± 0.001 s –1 ) and Michaelis constant K m (21 ± 1 μM). Such results confirmed the consistency of our kinetic data with those previously reported.…”

Section: Resultsmentioning

confidence: 58%

Extended Mechanism of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis: 1000-fold Higher Catalytic Activity than That of Clinically Used Alteplase

et al. 2022

View full text Add to dashboard Cite

The plasminogen activator staphylokinase is a fibrin-specific thrombolytic biomolecule and an attractive target for the development of effective myocardial infarction and stroke therapy. To engineer the protein rationally, a detailed understanding of the biochemical mechanism and limiting steps is essential. Conventional fitting to equations derived on the basis of simplifying approximations may be inaccurate for complex mechanisms such as that of staphylokinase. We employed a modern numerical approach of global kinetic data analysis whereby steady-state kinetics and binding affinity data sets were analyzed in parallel. Our approach provided an extended, revised understanding of the staphylokinase mechanism without simplifying approximations and determined the value of turnover number k cat of 117 s −1 that was 10000-fold higher than that reported in the literature. The model further showed that the rate-limiting step of the catalytic cycle is binding of staphylokinase to plasmin molecules, which occurs via an induced-fit mechanism. The overall staphylokinase effectivity is further influenced by the formation of an inactive staphylokinase•plasminogen complex. Here, we describe a quick and simplified guide for obtaining reliable estimates of key parameters whose determination is critical to fully understand the staphylokinase catalytic functionality and define rational strategies for its engineering. Our study provides an interesting example of how a global numerical analysis of kinetic data can be used to better understand the mechanism and limiting factors of complex biochemical processes. The high catalytic activity of staphylokinase (more than 1000-fold higher than that of the clinically used drug alteplase) determined herein makes this thrombolytic agent a very attractive target for further engineering.

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“…N -glycosylated variants of factor VII propeptides prolong its half-life significantly, while corresponding variants of factor IX can also play a role in the treatment of hemophilia [71,72]. Regulation of the fibrinolytic plasmin depends on differential N - and O -glycosylation, which alters the structure significantly [73,74]. In addition, both the tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) possess N - and O -glycans, whose roles have been partially elucidated [75,76].…”

Section: Glycosylated Proteasesmentioning

confidence: 99%

Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases

Goettig

2016

IJMS

100

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Posttranslational modifications are an important feature of most proteases in higher organisms, such as the conversion of inactive zymogens into active proteases. To date, little information is available on the role of glycosylation and functional implications for secreted proteases. Besides a stabilizing effect and protection against proteolysis, several proteases show a significant influence of glycosylation on the catalytic activity. Glycans can alter the substrate recognition, the specificity and binding affinity, as well as the turnover rates. However, there is currently no known general pattern, since glycosylation can have both stimulating and inhibiting effects on activity. Thus, a comparative analysis of individual cases with sufficient enzyme kinetic and structural data is a first approach to describe mechanistic principles that govern the effects of glycosylation on the function of proteases. The understanding of glycan functions becomes highly significant in proteomic and glycomic studies, which demonstrated that cancer-associated proteases, such as kallikrein-related peptidase 3, exhibit strongly altered glycosylation patterns in pathological cases. Such findings can contribute to a variety of future biomedical applications.

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The role of carbohydrate side chains of plasminogen in its activation by staphylokinase

Cited by 18 publications

References 34 publications

Extended Mechanism and Rate-Limiting Step of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis

Extended Mechanism and Rate-Limiting Step of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis

Extended Mechanism of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis: 1000-fold Higher Catalytic Activity than That of Clinically Used Alteplase

Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases

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