Hydrogen Bonding to Active-Site Histidine in Peptidyl Boronic Acid Inhibitor Complexes of Chymotrypsin and Subtilisin:  Proton Magnetic Resonance Assignments and H/D Fractionation

Bao, Donghui; Huskey, W. Phillip; Kettner, and Charles; Jordan, Frank

doi:10.1021/ja990180g

Cited by 44 publications

(63 citation statements)

References 51 publications

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“…It has also been observed before in ␣-chymotrypsin at low pH (24,25) and in an ␣-chymotrypsin inhibitor complex with a trifluoromethyl ketone inhibitor (25). The signal at ϳ13.0 ppm has been assigned to the N ⑀2 proton of the imidazolium ring of histidine 57 of ␣-chymotrypsin (24).…”

Section: H Nmr Of the Hydrogen-bonded Protons Of ␣-Chymotrypsin And Osupporting

confidence: 63%

13C and 1H NMR Studies of Ionizations and Hydrogen Bonding in Chymotrypsin-Glyoxal Inhibitor Complexes

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Cosgrove

Rogers

et al. 2007

Journal of Biological Chemistry

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Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-AlaPhe-glyoxal have both been shown to be inhibitors of ␣-chymotrypsin with minimal K i values of 19 and 344 nM, respectively, at neutral pH. These K i values increased at low and high pH with pK a values of ϳ4.0 and ϳ10.5, respectively. By using surface plasmon resonance, we show that the apparent association rate constant for Z-Ala-Pro-Phe-glyoxal is much lower than the value expected for a diffusion-controlled reaction.13 C NMR has been used to show that at low pH the glyoxal keto carbon is sp 3 -hybridized with a chemical shift of ϳ100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of ϳ91.6 ppm. The signal at ϳ100.7 ppm is assigned to the hemiketal formed between the hydroxy group of serine 195 and the keto carbon of the glyoxal. In a slow exchange process controlled by a pK a of ϳ4.5, the aldehyde carbon dehydrates to give a signal at ϳ205.5 ppm and the hemiketal forms an oxyanion at ϳ107.0 ppm. At higher pH, the re-hydration of the glyoxal aldehyde carbon leads to the signal at 107 ppm being replaced by a signal at 104 ppm (pK a ϳ9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-AlaAla-Phe-glyoxal to ␣-chymotrypsin at 4 and 25°C, 1 H NMR is used to show that the binding of these glyoxal inhibitors raises the pK a value of the imidazolium ion of histidine 57 to a value of >11 at both 4 and 25°C. We discuss the mechanistic significance of these results, and we propose that it is ligand binding that raises the pK a value of the imidazolium ring of histidine 57 allowing it to enhance the nucleophilicity of the hydroxy group of the active site serine 195 and lower the pK a value of the oxyanion forming a zwitterionic tetrahedral intermediate during catalysis.Specific substrate-derived glyoxal inhibitors have been shown to be potent inhibitors of the serine proteinases (1-4). Z 4 -Ala-Pro-Phe-glyoxal is an extremely potent reversible inhibitor of ␦-chymotrypsin with an apparent disassociation constant of 25 Ϯ 8 nM at pH 7.0 (1).The ␣-keto carbon of the glyoxal inhibitor is expected to occupy the same position as the carbonyl carbon of a substrate, and it has been shown that it is bound as a tetrahedral adduct, which should closely resemble the tetrahedral intermediate formed during substrate catalysis (1). By using 13 C NMR, it has been shown that ␦-chymotrypsin (1) and subtilisin (2) reduce the oxyanion pK a by ϳ6 and ϳ8 pK a units, respectively. It has been estimated that hydrogen bonding in the oxyanion hole will only reduce the oxyanion pK a by ϳ1.3 pK a units (1). This is consistent with the fact that hydrogen bonding is expected to be effective in both water and in the oxyanion hole, and so it should not reduce the oxyanion pK a to a value lower than that expected in water. This has led to the conclusion that hydrogen bonding in the oxyanion hole only has a minor role in lowering the oxyanion pK a (5-7). However, it has been proposed that substrate binding raises the pK a of the imidazolium ion of the active site histidine enabling ...

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Section: H Nmr Of the Hydrogen-bonded Protons Of ␣-Chymotrypsin And Osupporting

confidence: 63%

13C and 1H NMR Studies of Ionizations and Hydrogen Bonding in Chymotrypsin-Glyoxal Inhibitor Complexes

Spink

Cosgrove

Rogers

et al. 2007

Journal of Biological Chemistry

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“…and ~13.1 p.p.m. have been observed in chymotrypsin-glyoxal inhibitor complexes [21] and in chymotrypsintrifluoromethyl ketone inhibitor complexes [29]. The signals at 13.1 p.p.m.…”

Section: H-nmr Of the The Hydrogen Bonded Protons Of The Z-ala-ala-mentioning

confidence: 92%

Oxyanion and tetrahedral intermediate stabilisation by subtilisin: Detection of a new tetrahedral adduct

Howe

Rogers

Hewage

et al. 2009

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…A unique signal appears in high-resolution 1 H NMR spectra at low field between 14 and 21 ppm, in many cases of transition-state-analog adducts of enzymes with inhibitors that form tetrahedral intermediates. (33–45) The low-field signals can also be observed with some native enzymes at pH below 6. The phenomena have been interpreted as the presence of a short-strong-hydrogen bond (SSHB) at the active site of the enzyme.…”

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confidence: 99%

Proton Bridging in the Interactions of Thrombin with Small Inhibitors

et al. 2009

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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.

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Hydrogen Bonding to Active-Site Histidine in Peptidyl Boronic Acid Inhibitor Complexes of Chymotrypsin and Subtilisin: Proton Magnetic Resonance Assignments and H/D Fractionation

Cited by 44 publications

References 51 publications

13C and 1H NMR Studies of Ionizations and Hydrogen Bonding in Chymotrypsin-Glyoxal Inhibitor Complexes

13C and 1H NMR Studies of Ionizations and Hydrogen Bonding in Chymotrypsin-Glyoxal Inhibitor Complexes

Oxyanion and tetrahedral intermediate stabilisation by subtilisin: Detection of a new tetrahedral adduct

Proton Bridging in the Interactions of Thrombin with Small Inhibitors

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