Redesigning the monovalent cation specificity of an enzyme

Prasad, Swati; Wright, Kelly J.; Roy, Dolly Banerjee; Bush, Leslie A.; Cantwell, Angelene M.; Di, Enrico

doi:10.1073/pnas.2333109100

Cited by 57 publications

(103 citation statements)

References 59 publications

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“…We specifically sought to explain the molecular nature of the perturbation affecting a residue located >20 Å away from residues of the catalytic triad, the S1 pocket or the Na + site. Binding of Na + to thrombin elicits a 10-15% increase in intrinsic fluorescence [13,16,21,22] that is contributed by changes in the environment of all nine Trp residues of the enzyme distributed over the entire surface up to 35 Å away from the bound cation [16]. The fluorescence increase can be decomposed into a fast phase due to Na + binding to the E form to generate E:Na + and a slow phase due to the interconversion of the Na + -free forms E* and E [16,23].…”

Section: Resultsmentioning

confidence: 99%

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Role of the A chain in thrombin function

Papaconstantinou

Bah

2008

Cell. Mol. Life Sci.

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The A chain of thrombin is covalently linked to the catalytic B chain but is separate from any known epitope for substrate recognition. In this study we present the results of the Ala replacement of 12 charged residues controlling the stability of the A chain and its interaction with the B chain. Residues Arg4 and Glu8 play a significant role in substrate recognition, even though they are located >20 Å away from residues of the catalytic triad, the primary specificity pocket and the Na + site. The R4A mutation causes significant perturbation of Na + binding, fibrinogen clotting and PAR1 cleavage, but modest reduction of protein C activation in the presence of thrombomodulin. These findings challenge our current paradigm of thrombin structure-function relations focused exclusively on the properties of the catalytic B chain, and explain why certain naturally occurring mutations of the A chain cause serious bleeding.

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Section: Resultsmentioning

confidence: 99%

“…Site-directed mutagenesis of human thrombin was performed as described [11][12][13] using the Quik-Change site-directed mutagenesis kit from Stratagene (La Jolla, CA) in an HPC4-modified pNUT expression vector containing the human prethrombin-1 gene. Thrombin mutants were expressed in baby h amsterkidney cells.…”

Section: Methodsmentioning

confidence: 99%

Role of the A chain in thrombin function

Papaconstantinou

Bah

2008

Cell. Mol. Life Sci.

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“…Efforts continue to assess exactly why an enzyme chooses one of these monovalent cations over another. Strategies that successfully change monovalent cation specificity do not automatically lead to preservation of any cation-dependent allosteric events (1,5). In PNAS, Rana et al (6) delve into this unresolved issue in their article, "Redesigning allosteric activation in an enzyme."…”

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confidence: 99%

“…With this anticoagulant, both Na + and K + contribute comparable enhancement to catalytic activity (5,15). The 186 and the 220 loops of thrombin were replaced with the complementary ones for PC.…”

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confidence: 99%

Switching cation-binding loops paves the way for redesigning allosteric activation

Maurer¹

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Glu-217 makes polar contacts with Lys-224 and Thr-172, which helps to stabilize the intervening 220-loop in the Na ϩ site. The ion pair between Arg-187 and Asp-222 latches the 186-loop onto the 220-loop to stabilize the Na ϩ site and the pore of entry of the cation to its binding site (11). Tyr-225 plays a crucial role in determining the Na ϩ -dependent allosteric nature of serine proteases (4) by allowing the correct orientation of the backbone oxygen of residue 224 (12), which contributes directly to the coordination of Na ϩ .…”

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confidence: 99%

Murine Thrombin Lacks Na+ Activation but Retains High Catalytic Activity

Bush

Nelson

2006

Journal of Biological Chemistry

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Human thrombin utilizes Na؉ as a driving force for the cleavage of substrates mediating its procoagulant, prothrombotic, and signaling functions. Murine thrombin has Asp-222 in the Na ؉ binding site of the human enzyme replaced by Lys. The charge reversal substitution abrogates Na ؉ activation, which is partially restored with the K222D mutation, and ensures high activity even in the absence of Na ؉ . This property makes the murine enzyme more resistant to Thrombin is a Na ϩ -activated serine protease (1) that is responsible for the progression and regulation of blood coagulation (2). As in other monovalent cation-activated enzymes (3), the role of Na ϩ in thrombin function is to lower energy barriers for substrate binding in the ground and transition states so that physiologic substrates like PAR1 and fibrinogen can be hydrolyzed efficiently during platelet activation and formation of a blood clot. Other members of the vitamin K-dependent family of clotting proteases to which thrombin belongs are endowed with Na ϩ activation (4). In fact, the activity of activated protein C (5) and coagulation factors VIIa (6), IXa (7), and Xa (8) is influenced significantly by the presence of Na ϩ .Details of the molecular mechanism of Na ϩ activation in thrombin have emerged recently from mutagenesis and structural analysis (9). Na ϩ binding is severely compromised (Ͼ30-fold increase in K d ) upon mutation of Asp-189, Glu-217, Asp-222, and Tyr-225. Asp-189 fixes the orientation of one of the four water molecules ligating Na ϩ and provides an important link between the Na ϩ site and the P1 residue of substrate (10). Glu-217 makes polar contacts with Lys-224 and Thr-172, which helps to stabilize the intervening 220-loop in the Na ϩ site. The ion pair between Arg-187 and Asp-222 latches the 186-loop onto the 220-loop to stabilize the Na ϩ site and the pore of entry of the cation to its binding site (11). Tyr-225 plays a crucial role in determining the Na ϩ -dependent allosteric nature of serine proteases (4) by allowing the correct orientation of the backbone oxygen of residue 224 (12), which contributes directly to the coordination of Na ϩ . The side chain of Tyr-225 also secures the integrity of the water channel embedding the primary specificity pocket (12), and the backbone around Tyr-225 is oriented like the selectivity filter of the KcsA K ϩ channel (3, 13). Of these four residues, Glu-217 and Tyr-225 are conserved in thrombin from all species sequenced to date, from hagfish to human (14). Asp-189 is a Ser in the sturgeon, and the D189S mutant of human thrombin has impaired Na ϩ binding and substrate recognition (10). Asp-222 is the least conserved residue among the four. It is Ser in the sturgeon, Lys in the mouse, and Asn in the rat (14). The substitution in the mouse is particularly interesting, as it involves a charge reversal in the 220-loop that has the potential to destabilize the Na ϩ binding environment.Does the presence of Lys-222 in murine thrombin preclude Na However, the mutant has functional properties interm...

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Redesigning the monovalent cation specificity of an enzyme

Cited by 57 publications

References 59 publications

Role of the A chain in thrombin function

Role of the A chain in thrombin function

Switching cation-binding loops paves the way for redesigning allosteric activation

Murine Thrombin Lacks Na+ Activation but Retains High Catalytic Activity

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