Proton Inventory Studies of α-Thrombin-Catalyzed Reactions of Substrates with Selected P and P‘ Sites

Enyedy, Edith J.; Kovach, Ildiko M.

doi:10.1021/ja0320166

Cited by 20 publications

(67 citation statements)

References 53 publications

(109 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Based on the Ser-His dyad action in serine peptidases hydrolysis (17), the Glu 136 proton could be transferred to an acceptor atom as the substrate bound to enzyme. The decrease in k cat /K m obtained in the presence of D 2 O, similar to that observed with serine peptidases (18,33,40), supports this view. The extreme low pK value expected for such a acceptor residue suggests a carboxylate nature.…”

supporting

confidence: 81%

Studies on the Catalytic Mechanism of a Glutamic Peptidase

Kondo

Okamoto

Santos

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Scytalidoglutamic peptidase (SGP) is the prototype of fungal glutamic peptidases that are characteristically pepstatin insensitive. These enzymes have a unique catalytic dyad comprised of Gln 53 and Glu 136 that activate a bound water molecule for nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond. The hydrolysis by SGP at peptide bonds with proline in the P 1 position is a rare event among peptidases that we investigated using the series of fluorescence resonance energy transfer peptides, Abz-KLXPSKQ-EDDnp, compared with the series Abz-KLXSSKQ-EDDnp. The preference observed in these two series for Phe and His over Leu, Ile, Val, Arg, and Lys, seems to be related to the structure of the S 1 subsite of SGP. These results and the pH profiles of SGP activity showed that its S 1 subsite can accommodate the benzyl group of Phe at pH 4 as well as the positively charged imidazolium group of His. In the pH range 2 to 7, SGP maintains its structure and activity, but at pH 8 or higher it is irreversibly denatured. The intrinsic fluorescence of the Trp residues of SGP were sensitive to the titration of carboxyl groups having low pK values; this can be attributed to the buried Asp 57 and/or Asp 43 as described in SGP three-dimensional structure. The solvent kinetic isotope effects and the proton inventory experiments support a mechanism for the glutamic peptidase SGP that involves the nucleophilic attack of the general base (Glu 136 ) activated water, and establish a fundamental role of the S 1 subsite interactions in promoting catalysis.The recently established glutamic peptidase family (Family G1 in MEROPS, also known as the Eqolisins) is a novel group of acid peptidases having structures and mechanistic features distinct from the canonical peptidase families (1, 2), and so far, found only in the filamentous fungi (3) where they play key roles in fungal growth (4). Glutamic peptidases were first differentiated from aspartic peptidases as being insensitive to pepstatin and the absence of sequence similarity to the well characterized pepsin-like and retroviral aspartic peptidases (5, 6).The Scytalidoglutamic peptidase (SGP), 2 isolated from the wood-degrading fungus Scytalidium lignicolum, is the prototype of glutamic peptidases. Gln 53 and Glu 136 (in SGP numbering) were proposed to participate in the catalytic process because the single point mutants E136A, Q53A, and Q53E lost both the autoprocessing and the enzymatic activities (7). Sitedirected mutagenesis studies on Aspergillus niger glutamic peptidase established that these same residues constitute the catalytic dyad in this peptidase (8). The molecular structure of SGP was determined in its unbound native form, in complex with hydrolytic products and transition state peptide analogues (1, 9, 10). SGP has a unique ␤-strand tertiary structure among the known peptidases, and each layer of the sandwich is formed by seven antiparallel ␤-sheets. The catalytic residues Glu 136 and Gln 53 are both located in the same layer of the sandwich structure...

show abstract

supporting

confidence: 81%

Studies on the Catalytic Mechanism of a Glutamic Peptidase

Kondo

Okamoto

Santos

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The difference between these residues in the pK a was thought to be critical to their role as electrostatic modulators. For the serine protease thrombin, the inverse SIE observed is associated with a conformational change to release P 1 (40). By either mechanism, to affect P 1 release, the ester cleavage reaction cannot be facilitated by an analogous enzymesubstrate interaction.…”

Section: Discussionmentioning

confidence: 99%

Steady-State and Pre-Steady-State Kinetic Evaluation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CL^pro Cysteine Protease: Development of an Ion-Pair Model for Catalysis

et al. 2008

View full text Add to dashboard Cite

Severe acute respiratory syndrome (SARS) was a worldwide epidemic caused by a coronavirus that has a cysteine protease (3CLpro) essential to its life cycle. Steady-state and pre-steady-state kinetic methods were used with highly active 3CLpro to characterize the reaction mechanism. We show that 3CLpro has mechanistic features common and disparate to the archetypical proteases papain and chymotrypsin. The kinetic mechanism for 3CLpro-mediated ester hydrolysis, including the individual rate constants, is consistent with a simple double displacement mechanism. The pre-steady-state burst rate was independent of ester substrate concentration indicating a high commitment to catalysis. When homologous peptidic amide and ester substrates were compared, a series of interesting observations emerged. Despite a 2000-fold difference in nonenzymatic reactivity, highly related amide and ester substrates were found to have similar kinetic parameters in both the steady-state and pre-steady-state. Steady-state solvent isotope effect (SIE) studies showed an inverse SIE for the amide but not ester substrates. Evaluation of the SIE in the pre-steady-state revealed normal SIEs for both amide and ester burst rates. Proton inventory (PI) studies on amide peptide hydrolysis were consistent with two proton-transfer reactions in the transition state while the ester data was consistent with a single proton-transfer reaction. Finally, the pH-inactivation profile of 3CLpro with iodoacetamide is indicative of an ion-pair mechanism. Taken together, the data are consistent with a 3CLpro mechanism that utilizes an "electrostatic" trigger to initiate the acylation reaction, a cysteine-histidine catalytic dyad ion pair, an enzyme-facilitated release of P1, and a general base-catalyzed deacylation reaction.

show abstract

“…Thrombin concentrations, [E], were then calculated from the V max values using k cat = 95 ± 20 s −1 in pH 8.0, 0.02 M Tris buffer at 25.0 ± 0.1 °C. (22)…”

Section: Methodsmentioning

confidence: 99%

“…This notion is supported by the observed large solvent deuterium isotope effects in the hydrolysis of many thrombin substrates. (22;28) These isotope effects between 2.5 and 3.5 are most likely primary effects, meaning that protons are transferred in the rate-determining step of the hydrolysis reaction. The origin of this difference between proton and deuteron transfer at the transition state lies in the loss of the difference in zero-point energies existing in the ground state vibration of H/D in bonds to heavy atoms.…”

mentioning

confidence: 99%

Proton Bridging in the Interactions of Thrombin with Small Inhibitors

et al. 2009

Self Cite

View full text Add to dashboard Cite

Thrombin is the pivotal serine protease enzyme in the blood cascade system. Phe-Pro-Arg-chloromethylketone (PPACK), phosphate and phosphonate ester inhibitors form a covalent bond with the active-site Ser of thrombin. PPACK, a mechanism-based inhibitor, and the phosphate/phosphonate esters form adducts that mimic intermediates formed in reactions catalyzed by thrombin. Therefore, the dependence of the inhibition of human α-thrombin on the concentration of these inhibitors, pH, and temperature was investigated. The second-order rate constant, ki/Ki, and the inhibition constant, Ki, for inhibition of human α-thrombin by PPACK are (1.1 ± 0.2) × 107 M−1 s−1 and (2.4 ± 1.3) × 10−8 M at pH 7.00 in 0.05 M phosphate buffer, 0.15 M NaCl, and 25.0 ± 0.1˚C, and in good agreement with previous reports. The activation parameters at pH 7.00, 0.05M phosphate buffer 0.15 M NaCl, are ΔH‡ = 10.6 ± 0.7 kcal/mol and ΔS‡ = 9 ± 2 cal/mol deg. The pH dependence of the second-order rate constants of inhibition is bell shaped. Values of pKa1 and pKa2 are 7.3 ± 0.2 and 8.8 ± 0.3, respectively, at 25.0 ± 0.1 °C. A phosphate and a phosphonate ester inhibitor gave higher values, 7.8 and 8.0, for pKa1 and 9.3 and 8.6 for pKa2. They inhibit thrombin over six orders of magnitude less efficiently than PPACK does. The deuterium solvent isotope effect for the second-order rate constant at pH 7.0 and 8.3 at 25.0 ± 0.1°C is unity within experimental error in all three cases, indicating the absence of proton transfer in the rate-determining step for the association of thrombin with the inhibitors. But in a 600 MHz 1H NMR spectrum of the inhibition adduct at pH 6.7 and 30 °C, a peak at 18.10 ppm with respect to TSP appears with PPACK, which is absent in the 1H NMR spectrum of a solution of the enzyme between pH 5.3–8.5. The peak at low field is an indication of the presence an SSHB at the active site in the adduct. The deuterium isotope effect on this hydrogen bridge is 2.2 ± 0.2 (ϕ = 0.45). The presence of an SSHB is also established with a signal at 17.34 ppm for a dealkylated phosphate adduct of thrombin.

show abstract

Proton Inventory Studies of α-Thrombin-Catalyzed Reactions of Substrates with Selected P and P‘ Sites

Cited by 20 publications

References 53 publications

Studies on the Catalytic Mechanism of a Glutamic Peptidase

Studies on the Catalytic Mechanism of a Glutamic Peptidase

Steady-State and Pre-Steady-State Kinetic Evaluation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CL^pro Cysteine Protease: Development of an Ion-Pair Model for Catalysis

Proton Bridging in the Interactions of Thrombin with Small Inhibitors

Contact Info

Product

Resources

About

Proton Inventory Studies of α-Thrombin-Catalyzed Reactions of Substrates with Selected P and P‘ Sites

Cited by 20 publications

References 53 publications

Studies on the Catalytic Mechanism of a Glutamic Peptidase

Studies on the Catalytic Mechanism of a Glutamic Peptidase

Steady-State and Pre-Steady-State Kinetic Evaluation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CLpro Cysteine Protease: Development of an Ion-Pair Model for Catalysis

Proton Bridging in the Interactions of Thrombin with Small Inhibitors

Contact Info

Product

Resources

About

Steady-State and Pre-Steady-State Kinetic Evaluation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CL^pro Cysteine Protease: Development of an Ion-Pair Model for Catalysis