Screening of the active site from water by the incoming ligand triggers catalysis and inhibition in serine proteases

Michael Shokhen; Netaly Khazanov; Amnon Albeck

doi:10.1002/prot.21727

Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex

Scientific Reports ◽

10.1038/s41598-021-88432-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Riley B. Peacock ◽

Taylor McGrann ◽

Marco Tonelli ◽

Elizabeth A. Komives

Keyword(s):

Active Site ◽

Serine Proteases ◽

Protein C ◽

Catalytic Mechanism ◽

Bond Cleavage ◽

Peptide Bond ◽

Peptide Bond Cleavage ◽

Exchange Mass Spectrometry ◽

Catalytically Active ◽

Hydrogen Deuterium

AbstractSerine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-β-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced μs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased μs-ms dynamics in a β-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two β-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

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Probing catalytic rate enhancement during intramembrane proteolysis

Biological Chemistry ◽

10.1515/hsz-2016-0124 ◽

2016 ◽

Vol 397 (9) ◽

pp. 907-919 ◽

Cited By ~ 4

Author(s):

Elena Arutyunova ◽

Cameron C. Smithers ◽

Valentina Corradi ◽

Adam C. Espiritu ◽

Howard S. Young ◽

...

Keyword(s):

Active Site ◽

Serine Proteases ◽

Homology Model ◽

Intramembrane Proteolysis ◽

Oxyanion Hole ◽

Rate Enhancement ◽

Transition State Stabilization ◽

The Family ◽

Catalytic Dyad

Abstract Rhomboids are ubiquitous intramembrane serine proteases involved in various signaling pathways. While the high-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed an active site comprised of a serine-histidine dyad and an extensive oxyanion hole, the molecular details of rhomboid catalysis were unclear because substrates are unknown for most of the family members. Here we used the only known physiological pair of AarA rhomboid with its psTatA substrate to decipher the contribution of catalytically important residues to the reaction rate enhancement. An MD-refined homology model of AarA was used to identify residues important for catalysis. We demonstrated that the AarA active site geometry is strict and intolerant to alterations. We probed the roles of H83 and N87 oxyanion hole residues and determined that substitution of H83 either abolished AarA activity or reduced the transition state stabilization energy (ΔΔG‡) by 3.1 kcal/mol; substitution of N87 decreased ΔΔG‡ by 1.6–3.9 kcal/mol. Substitution M154, a residue conserved in most rhomboids that stabilizes the catalytic general base, to tyrosine, provided insight into the mechanism of nucleophile generation for the catalytic dyad. This study provides a quantitative evaluation of the role of several residues important for hydrolytic efficiency and oxyanion stabilization during intramembrane proteolysis.

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Short hydrogen bonds in the catalytic mechanism of serine proteases

Journal of the Serbian Chemical Society ◽

10.2298/jsc0804393l ◽

2008 ◽

Vol 73 (4) ◽

pp. 393-403 ◽

Cited By ~ 5

Author(s):

Vladimir Leskovac ◽

Svetlana Trivic ◽

Draginja Pericin ◽

Mira Popovic ◽

Julijan Kandrac

Keyword(s):

Hydrogen Bond ◽

Hydrogen Bonds ◽

Active Site ◽

Serine Proteases ◽

Active Sites ◽

Catalytic Mechanism ◽

Data Bank ◽

Ground State Structure ◽

Low Barrier Hydrogen Bonds ◽

Short Hydrogen Bonds

The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78-1.28 ? revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 ? long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short "low-barrier" hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the "low-barrier hydrogen bond" hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 ? long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the "low-barrier hydrogen bond" hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

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PREDICTION OF THE THREE-DIMENSIONAL STRUCTURE OF THE ENZYMATIC PART OF T-PA

10.1055/s-0038-1644407 ◽

1987 ◽

Author(s):

A Heckel ◽

K M Hasselbach

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Active Site ◽

Serine Proteases ◽

Three Dimensional ◽

Amino Acid Sequences ◽

Dimensional Structure ◽

Salt Bridges ◽

Three Dimensional Structure ◽

Insertions And Deletions

Up to now the three-dimensional structure of t-PA or parts of this enzyme is unknown. Using computer graphical methods the spatial structure of the enzymatic part of t-PA is predicted on the hypothesis, the three-dimensional backbone structure of t-PA being similar to that of other serine proteases. The t-PA model was built up in three steps:1) Alignment of the t-PA sequence with other serine proteases. Comparison of enzyme structures available from Brookhaven Protein Data Bank proved elastase as a basis for modeling.2) Exchange of amino acids of elastase differing from the t-PA sequence. The replacement of amino acids was performed such that backbone atoms overlapp completely and side chains superpose as far as possible.3) Modeling of insertions and deletions. To determine the spatial arrangement of insertions and deletions parts of related enzymes such as chymotrypsin or trypsin were used whenever possible. Otherwise additional amino acid sequences were folded to a B-turn at the surface of the proteine, where all insertions or deletions are located. Finally the side chain torsion angles of amino acids were optimised to prevent close contacts of neigh bouring atoms and to improve hydrogen bonds and salt bridges.The resulting model was used to explain binding of arginine 560 of plasminogen to the active site of t-PA. Arginine 560 interacts with Asp 189, Gly 19 3, Ser 19 5 and Ser 214 of t-PA (chymotrypsin numbering). Furthermore interaction of chromo-genic substrate S 2288 with the active site of t-PA was studied. The need for D-configuration of the hydrophobic amino acid at the N-terminus of this tripeptide derivative could be easily explained.

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Active site-specific immunoassays

Blood ◽

10.1182/blood.v76.4.755.755 ◽

1990 ◽

Vol 76 (4) ◽

pp. 755-766 ◽

Cited By ~ 15

Author(s):

KG Mann ◽

EB Williams ◽

S Krishnaswamy ◽

W Church ◽

A Giles ◽

...

Keyword(s):

Active Site ◽

Serine Proteases ◽

Solid Phase ◽

Activated Protein C ◽

Factor Xa ◽

Immune Recognition ◽

Factor X ◽

Zymogen Activation ◽

Prothrombinase Complex ◽

Active Site Histidine

Abstract This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl- epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.

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The Herpesvirus Proteases as Targets for Antiviral Chemotherapy

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020001100101 ◽

2000 ◽

Vol 11 (1) ◽

pp. 1-22 ◽

Cited By ~ 85

Author(s):

Lloyd Waxman ◽

Paul L Darke

Keyword(s):

Serine Protease ◽

Active Site ◽

Serine Proteases ◽

Catalytic Triad ◽

Active Site Residues ◽

Virus Family ◽

Single Domain Protein ◽

Inhibitor Discovery ◽

Antiviral Chemotherapy ◽

Simplex Virus

Viruses of the family Herpesviridae are responsible for a diverse set of human diseases. The available treatments are largely ineffective, with the exception of a few drugs for treatment of herpes simplex virus (HSV) infections. For several members of this DNA virus family, advances have been made recently in the biochemistry and structural biology of the essential viral protease, revealing common features that may be possible to exploit in the development of a new class of anti-herpesvirus agents. The herpesvirus proteases have been identified as belonging to a unique class of serine protease, with a Ser-His-His catalytic triad. A new, single domain protein fold has been determined by X-ray crystallography for the proteases of at least three different herpesviruses. Also unique for serine proteases, dimerization has been shown to be required for activity of the cytomegalovirus and HSV proteases. The dimerization requirement seriously impacts methods needed for productive, functional analysis and inhibitor discovery. The conserved functional and catalytic properties of the herpesvirus proteases lead to common considerations for this group of proteases in the early phases of inhibitor discovery. In general, classical serine protease inhibitors that react with active site residues do not readily inactivate the herpesvirus proteases. There has been progress however, with activated carbonyls that exploit the selective nucleophilicity of the active site serine. In addition, screening of chemical libraries has yielded novel structures as starting points for drug development. Recent crystal structures of the herpesvirus proteases now allow more direct interpretation of ligand structure—activity relationships. This review first describes basic functional aspects of herpesvirus protease biology and enzymology. Then we discuss inhibitors identified to date and the prospects for their future development.

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Comparative Study of the Active Site Caging of Serine Proteases: Thrombin and Factor Xa†

Biochemistry ◽

10.1021/bi0106078 ◽

2002 ◽

Vol 41 (6) ◽

pp. 2002-2013 ◽

Cited By ~ 12

Author(s):

Jan Willem Thuring ◽

Hui Li ◽

Ned A. Porter

Keyword(s):

Comparative Study ◽

Active Site ◽

Serine Proteases ◽

Factor Xa

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Oxidative inactivation of plasmin and other serine proteases by copper and ascorbate

Blood ◽

10.1182/blood.v82.5.1522.1522 ◽

1993 ◽

Vol 82 (5) ◽

pp. 1522-1531 ◽

Cited By ~ 20

Author(s):

SE Lind ◽

JR McDonagh ◽

CJ Smith

Keyword(s):

Plasminogen Activator ◽

Active Site ◽

Serine Proteases ◽

Plasminogen Activators ◽

Single Chain ◽

Site Specific ◽

Oxidative Inactivation ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Specific Oxidation

Abstract Fibrin thrombi form at sites of injury, where leukocytes release a variety of oxidants. To determine whether oxidants might affect proteins of the fibrinolytic system, we examined the effects of various oxidants on plasmin. Plasmin was not inhibited by micromolar concentrations of hypochlorous acid, chloramine T, or H2O2. Neither Fe nor Cu affected plasmin alone or in the presence of H2O2. However, incubation of plasmin with 5 mumol/L Cu(I or II) in the presence of the reducing agent ascorbic acid resulted in a loss of its hydrolytic activity towards proteins as well as towards small synthetic substrates. The addition of EDTA, but not mannitol, prevented its inactivation. Inactivation was prevented by the addition of catalase and accelerated by hydrogen peroxide. Preincubation of plasmin with the competitive inhibitor alpha-N-acetyl-L-lysine methyl ester prevented inactivation by Cu(II) and ascorbate. These results together suggest site-specific oxidation of plasmin's active site. Treatment of the plasminogen activators tissue plasminogen activator and two-chain urokinase-type plasminogen activator, as well as trypsin, neutrophil elastase, and thrombin with Cu(II) and ascorbate resulted in a loss of their amidolytic and proteolytic activity, indicating the general susceptibility of serine proteases to this type of oxidation. Oxidation of the zymogens Glu-plasminogen and single-chain urokinase-type plasminogen activator by Cu(II) and ascorbate resulted in the failure of these molecules to generate active enzymes when treated with plasminogen activators or plasmin, respectively. The active site His residue may be the target of oxidative inactivation, as evidenced by the partial protection afforded plasmin by the addition of Zn(II), histidine, or the platinum derivative, platinum(II) (2,2′:6′,2″- terpyridine) chloride. Because platelets contain micromolar concentrations of Cu and leukocytes are rich in ascorbate, Cu-dependent site-specific oxidation might play a role in modulating proteolytic events and the life span of thrombi formed at sites of tissue injury.

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