The electrostatic fields in the active-site clefts of actinidin and papain.
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ABSTRACT:
SUBMITTER: Pickersgill RW
PROVIDER: S-EPMC1135062 | biostudies-other | 1988 Aug
REPOSITORIES: biostudies-other
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Project description:1. The pH-dependence of the second-order rate constant (k) for the reaction of actinidin (EC 3.4.22.14) with 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide was determined and the contributions to k of various hydronic states were evaluated. 2. The data were used to assess the consequences for transition-state geometry of providing P2/S2 hydrophobic contacts in addition to hydrogen-bonding opportunities in the S1-S2 intersubsite region. 3. The P2/S2 contacts (a) substantially improve enzyme-ligand binding, (b) greatly enhance the contribution to reactivity of the hydronic state bounded by pKa 3 (the pKa characteristic of the formation of catalytic-site-S-/-ImH+ state) and pKa 5 (a relatively minor contributor in reactions that lack the P2/S2 contacts), such that the major rate optimum occurs at pH 4 instead of at pH 2.8-2.9, and (c) reveal the kinetic influence of a pKa approx. 6.3 not hitherto observed in reactions of actinidin. 4. Possibilities for the interplay of electrostatic effects and binding interactions in both actinidin and papain (EC 3.4.22.2) are discussed.
| S-EPMC1138529 | biostudies-other
Project description:Vibrational Stark effect spectroscopy was used to measure electrostatic fields in the hydrophobic region of the active site of human aldose reductase (hALR2). A new nitrile-containing inhibitor was designed and synthesized, and the X-ray structure of its complex, along with cofactor NADP(+), with wild-type hALR2 was determined at 1.3 Å resolution. The nitrile is found to be in the proximity of T113, consistent with a hydrogen bond interaction. Two vibrational absorption peaks were observed at room temperature in the nitrile region when the inhibitor binds to wild-type hALR2, indicating that the nitrile probe experiences two different microenvironments, and these could be empirically separated into a hydrogen-bonded and non-hydrogen-bonded population by comparison with the T113A mutant, in which a hydrogen bond to the nitrile is not present. Classical molecular dynamics simulations based on the structure predict a double-peak distribution in protein electric fields projected along the nitrile probe. The interpretation of these two peaks as a hydrogen bond formation-dissociation process between the probe nitrile group and a nearby amino acid side chain is used to explain the observation of two IR bands, and the simulations were used to investigate the molecular details of this conformational change. Hydrogen bonding complicates the simplest analysis of vibrational frequency shifts as being due solely to electrostatic interactions through the vibrational Stark effect, and the consequences of this complication are discussed.
| S-EPMC3183354 | biostudies-literature
Project description:The leader proteinase (L(pro)) of foot-and-mouth-disease virus is an unusual papain-like cysteine proteinase. Synthesized without an N-terminal pro precursor region, it frees itself from the growing polypeptide chain by cleavage at its own C-terminus. It also possesses a unique electrostatic environment around the active site, essentially due to Asp(163), which orients the catalytic histidine residue, and Asp(164); the equivalent residues in papain are Asn(175) and Ser(176). The importance of these residues for L(pro) activity was examined by site-directed mutagenesis. Replacement of Asp(163) with asparagine reduced activity by five-fold towards a hexapeptide substrate and slightly delayed self-processing when expressed in rabbit reticulocyte lysates. However, no effect on the cleavage of the only known cellular substrate of L(pro), eukaryotic initiation factor 4GI (eIF4GI), was observed. In contrast, replacement of Asp(164) by either alanine, asparagine or lysine abrogated activity towards the hexapeptide. Furthermore, in all cases, the onset of both self-processing and eIF4GI cleavage were significantly delayed; the reaction rates were also diminished compared with those of the wild-type enzyme. The alanine-substituted enzyme was least affected, followed by those substituted with asparagine and lysine. The double mutant protein in which both aspartate residues were replaced by asparagine was most severely affected; it failed to complete either self-processing or eIF4GI cleavage within 3 h, compared with the 8 min required by the wild-type enzyme. Hence, we propose that the electrostatic charge of Asp(164), and to a lesser extent that of Asp(163), is extremely important for L(pro) to attain full activity upon synthesis.
| S-EPMC1222501 | biostudies-other
Project description:Papain was irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. The molecular weight of the inhibited enzyme was indistinguishable from that of papain itself, and no evidence of dimeric or oligomeric species was found. The optical-rotatory-dispersion curves of chloroacetone-inhibited papain and 1,3-dibromoacetone-inhibited papain were essentially similar. Amino acid analysis of the 1,3-dibromo[2-(14)C]acetone-inhibited enzyme and the performic acid-oxidized material clearly showed that a cysteine and histidine residue had been alkylated through the thiol and N-1 of the imidazole group respectively. These groups must therefore be within 5å of each other in the tertiary structure of papain. Possible mechanistic implications are briefly discussed.
| S-EPMC1198892 | biostudies-other
Project description:7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) and 7-(2'-hydroxyethylthio)-NBD (obtained from NBD chloride and mercaptoethanol) undergo a reversible spectral change in alkaline solution that depends respectively on a single apparent pK(a) 9.76 (at 25 degrees C) and 8.81 (at 32 degrees C). In acid solution however no spectral change was observed. NBD chloride reacts slowly with papain at pH7, but the rate of inhibition increases at lower pH and depends on an apparent pK(a) of 3.7 (at 35 degrees C), which has been tentatively assigned to the carboxyl group of aspartic acid-158. The spectral properties of NBD-papain indicate that the thiol group of cysteine-25 is the site of reaction. The intensity of the fluorescence-emission spectrum of NBD-papain depends on a single pK(a) of 4.2 (at 26.7 degrees C). The intensity of the fluorescence-emission spectrum of the mixed disulphide formed from papain and 7-(2'-mercaptoethylamino)-NBD (obtained from NBD chloride and cysteamine) depended on a single pK(a) of 3.94 in water and 3.89 in aq. 19.2% (v/v) dioxan (at 27 degrees C). This small change to lower pK(a) value in a medium of lower dielectric constant is characteristic of a cationic acid. The only acid of this type in the active-site region is the conjugate acid of histidine-159.
| S-EPMC1177757 | biostudies-other
Project description:Catechol-O-methyltransferase (COMT) is a model S-adenosyl-l-methionine (SAM) dependent methyl transferase, which catalyzes the methylation of catecholamine neurotransmitters such as dopamine in the primary pathway of neurotransmitter deactivation in animals. Despite extensive study, there is no consensus view of the physical basis of catalysis in COMT. Further progress requires experimental data that directly probes active site geometry, protein dynamics and electrostatics, ideally in a range of positions along the reaction coordinate. Here we establish that sinefungin, a fungal-derived inhibitor of SAM-dependent enzymes that possess transition state-like charge on the transferring group, can be used as a transition state analog of COMT when combined with a catechol. X-ray crystal structures and NMR backbone assignments of the ternary complexes of the soluble form of human COMT containing dinitrocatechol, Mg2+ and SAM or sinefungin were determined. Comparison and further analysis with the aid of density functional theory calculations and molecular dynamics simulations provides evidence for active site "compaction", which is driven by electrostatic stabilization between the transferring methyl group and "equatorial" active site residues that are orthogonal to the donor-acceptor (pseudo reaction) coordinate. We propose that upon catecholamine binding and subsequent proton transfer to Lys 144, the enzyme becomes geometrically preorganized, with little further movement along the donor-acceptor coordinate required for methyl transfer. Catalysis is then largely facilitated through stabilization of the developing charge on the transferring methyl group via "equatorial" H-bonding and electrostatic interactions orthogonal to the donor-acceptor coordinate.
| S-EPMC6503465 | biostudies-literature
Project description:Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these electric fields have proven difficult to quantify with standard experimental approaches. Using vibrational Stark effect spectroscopy, we found that the active site of the enzyme ketosteroid isomerase (KSI) exerts an extremely large electric field onto the C=O chemical bond that undergoes a charge rearrangement in KSI's rate-determining step. Moreover, we found that the magnitude of the electric field exerted by the active site strongly correlates with the enzyme's catalytic rate enhancement, enabling us to quantify the fraction of the catalytic effect that is electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomolecular systems.
| S-EPMC4668018 | biostudies-literature
Project description:A compound bound covalently to an enzyme active site can act either as a substrate if the covalent linkage is readily broken up by the enzyme or as an inhibitor if the bond dissociates slowly. We tracked the reactivity of such bonds associated with the rise of the resistance to penicillin G (PenG) in protein evolution from penicillin-binding proteins (PBPs) to TEM β-lactamases and with the development of avibactam (Avb) to overcome the resistance. We found that the ester linkage in PBP-PenG is resistant to hydrolysis mainly due to the small electric fields present in the protein active site. Conversely, the same linkage in the descendant TEM-PenG experiences large electric fields that stabilize the more charge-separated transition state and thus lower the free energy barrier to hydrolysis. Specifically, the electric fields were improved from -59 to -140 MV/cm in an ancient evolution dating back billions of years, contributing 5 orders of magnitude rate acceleration. This trend continues today in the nullification of newly developed antibiotic drugs. The fast linkage hydrolysis acquired from evolution is counteracted by the upgrade of PenG to Avb whose linkage escapes from the hydrolysis by returning to a low-field environment. Using the framework of electrostatic catalysis, the electric field, an observable from vibrational spectroscopy, provides a unifying physical metric to understand protein evolution and to guide the design of covalent drugs.
| S-EPMC10075085 | biostudies-literature
Project description:The peroxiredoxins (Prx) are ubiquitous peroxidases involved in important biological processes; however, details of their enzymatic mechanism remain elusive. To probe potential dynamics-function relationships, molecular dynamics simulations and electrostatic calculations were performed on the atypical 2-cysteine thiol peroxidase (Tpx) from Streptococcus pneumoniae and results compared to a previous study of a typical 2-cysteine Prx from Trypanosoma cruzi. The analyses indicate a commonality between both typical and atypical Prx: dynamic asymmetry. Asymmetry is observed in structure, fluctuations, and active site electrostatics. Key residues, including Glu150 and Phe153, play roles in the developing asymmetry; furthermore, in the atypical 2-Cys Tpx, Glu150 exhibits conformation fluctuations suggesting involvement in a proton shuttle. The existence of a pathway of connected residues appears to propagate the asymmetry. The commonality of asymmetry and coupling pathways in both typical and atypical Prxs suggests a driving force toward dimer asymmetry as a common feature that plays a functional role in creating one active site with a lower cysteine pK(a).
| S-EPMC3383837 | biostudies-literature
Project description:We have shown previously that the solvent-induced equilibrium unfolding mechanism of class Sigma glutathione S-transferase (GST) is strongly affected by ionic strength [Stevens, Hornby, Armstrong and Dirr (1998) Biochemistry 37, 15534-15541]. The protein is dimeric and has a hydrophilic subunit interface. Here we show that ionic strength alone has significant effects on the conformation of the protein, in particular at the active site. With the use of NaCl at up to 2 M under equilibrium conditions, the protein lost 60% of its catalytic activity and the single tryptophan residue per subunit became partly exposed. The effect was independent of protein concentration, eliminating the dissociation of the dimer as a possibility for the conformational changes. This was confirmed by size-exclusion HPLC. There was no significant change in the secondary structure of the protein according to far-UV CD data. Manual-mixing and stopped-flow kinetics experiments showed a slow single-exponential salt-induced change in protein fluorescence. For equilibrium and kinetics experiments, the addition of an active-site ligand (S-hexylglutathione) completely protected the protein from the ionic-strength-induced conformational changes. This suggests that the change occurs at or near the active site. Possible structural reasons for these novel effects are proposed, such as the flexibility of the alpha-helix 2 region as well as the hydrophilic subunit interface, highlighting the importance of electrostatic interactions in maintaining the structure of the active site of this GST.
| S-EPMC1220947 | biostudies-other