Active Site Zn Research Articles

Collaborative optimization and molecular docking exploration of novel ACE-inhibitory peptides from bovine milk by complex proteases hydrolysis

Food-originated angiotensin-I-converting enzyme (ACE)-inhibitory peptides to preserve hypertension are widely investigated over the past decade. Our research aims to discovery novel ACE-inhibitory peptides from bovine milk by couple of complex proteases (alcalase and protease). By means of response surface methodology with the conditions of pH 9.01, 61.81 °C and 6.5% ratio of enzyme to substrate, the hydrolysis model contributes to best-performing ACE-inhibitory activity of 85.02%. Through the further purification by consequent ultrafiltration, macroporous resin and gel chromatography, fraction G2-2 is eventually obtained with ACE-inhibitory activity as high as 92.7%. Two novel peptides of VLPVPQ and VAPFPE are identified by Q-Exactive LC–MS/MS. The molecular docking study further suggests that two novel peptides have good combinations of the S1 and S2 active site pockets and Zn(II) of ACE. Our study provides a fitted mathematical model to produce two novel milk-derived ACE-inhibitory peptides, potentially developing the functional foods, especially for hypertension therapy as initial treatment.

Interplay between β-lactamases and new β-lactamase inhibitors.

Resistance to β-lactam antibiotics in Gram-negative bacteria is commonly associated with production of β-lactamases, including extended-spectrum β-lactamases (ESBLs) and carbapenemases belonging to different molecular classes: those with a catalytically active serine and those with at least one active-site Zn2+ to facilitate hydrolysis. To counteract the hydrolytic activity of these enzymes, combinations of a β-lactam with a β-lactamase inhibitor (BLI) have been clinically successful. However, some β-lactam-BLI combinations have lost their effectiveness against prevalent Gram-negative pathogens that produce ESBLs, carbapenemases or multiple β-lactamases in the same organism. In this Review, descriptions are provided for medically relevant β-lactamase families and various BLI combinations that have been developed or are under development. Recently approved inhibitor combinations include the inhibitors avibactam and vaborbactam of the diazabicyclooctanone and boronic acid inhibitor classes, respectively, as new scaffolds for future inhibitor design.

Regulation of histone deacetylase 3 by metal cations and 10-hydroxy-2E-decenoic acid: Possible epigenetic mechanisms of queen-worker bee differentiation.

Histone deacetylases (HDACs) catalyze the hydrolysis of Ɛ-acetyl-lysine residues of histones. Removal of acetyl groups results in condensation of chromatin structure and repression of gene expression. Human class I, II, and IV HDACs are said to be zinc-dependent in that they require divalent zinc ions to catalyze the deacetylase reaction. HDACs are considered potential targets for the treatment of cancer due to their role in regulating transcription. They are also thought to play important roles in the development of organisms such as honey bees. The fatty acid, 10-hydroxy-2E-decenoic acid (10-HDA), which can account for up to 5% of royal jelly composition has been reported as an HDAC inhibitor. The crystal structure of the HDAC3:SMRT complex possesses two monovalent cations (MVCs) labeled as potassium with one MVC binding site near the active site Zn(II) and the second MVC binding site ≥20 Å from the active site Zn(II). We report here the inhibitory effects of excess Zn(II) on the catalytic activity of histone deacetylase 3 (HDAC3) bound to the deacetylase activating domain of nuclear receptor corepressor 2 (NCOR2). We also report the effects of varying concentrations of potassium ions where [K+] up to 10 mM increase HDAC3 activity with a maximum kcat/KM of approximately 80,000 M-1s-1 while [K+] above 10 mM inhibit HDAC3 activity. The inhibition constant (Ki) of 10-HDA was determined to be 5.32 mM. The regulatory effects of zinc, potassium, and 10-HDA concentration on HDAC3 activity suggest a strong correlation between these chemical species and epigenetic control over Apis mellifera caste differentiation among other control mechanisms.

Computational Studies on the Inhibitor Selectivity of Human JAMM Deubiquitinylases Rpn11 and CSN5.

Deubiquitinylases (DUBs) are highly specialized enzymes which are responsible for removal of covalently attached ubiquitin(s) from the targeted proteins. DUBs play an important role in maintaining the protein homeodynamics. Recently, DUBs have emerged as novel therapeutic targets in cancer, inflammation, diabetes, and neurodegeneration. Among the different families of DUBs, the metalloprotease group or JAB1/MOV34/MPR1 (JAMMs) proteases are unique in terms of catalytic mechanism. JAMMs exhibit a Zn2+-dependent deubiquitinylase activity. Within the JAMM family, deubiquitinylases Rpn11 and CSN5 are constituents of large bimolecular complexes, namely the 26S proteasome and COP9 signalosome (CSN), respectively. Rpn11 and CSN5 are potential drug targets in cancer and selective inhibitors of both proteins have been reported in the literature. However, the selectivity of JAMM inhibitors (capzimin for RPN11 and CSN5i-3 for CSN5) has not been structurally resolved yet. In the present work, we have explored the binding modes of capzimin and CSN5i-3 and rationalize their selectivity for Rpn11 and CSN5 targets. We found that capzimin interacts with the active site Zn+2 of Rpn11 in a bidentate manner and also interacts with the residues in the distal ubiquitin binding site. MD simulations studies and binding energy analysis revealed that the selective binding of the inhibitors can be only explained by the consideration of larger heterodimeric complexes of Rpn11 (Rpn8-Rpn11) and CSN5 (CSN5-CSN6). Simulation of these protein-protein complexes is necessary to avoid unrealistic large conformational changes. The selective binding of inhibitors is mainly governed by residues in the distal ubiquitin binding site. This study demonstrates that selective inhibitor binding design for Rpn11 and CSN5 JAMM proteases requires consideration of heterodimeric protein-protein target structures.

Class I Histone Deacetylase Inhibition by Tianeptinaline Modulates Neuroplasticity and Enhances Memory.

Through epigenetic and other regulatory functions, the histone deacetylase (HDAC) family of enzymes has emerged as a promising therapeutic target for central nervous system and other disorders. Here we report on the synthesis and functional characterization of new HDAC inhibitors based structurally on tianeptine, a drug used primarily to treat major depressive disorder (MDD) that has a poorly understood mechanism of action. Since the chemical structure of tianeptine resembles certain HDAC inhibitors, we profiled the in vitro HDAC inhibitory activity of tianeptine and demonstrated its ability to inhibit the lysine deacetylase activity of a subset of class I HDACs. Consistent with a model of active site Zn2+ chelation by the carboxylic acid present in tianeptine, newly synthesized analogues containing either a hydroxamic acid or ortho-aminoanilide exhibited increased potency and selectivity among the HDAC family. This in vitro potency translated to improved efficacy in a panel of high-content imaging assays designed to assess HDAC target engagement and functional effects on critical pathways involved in neuroplasticity in both primary mouse neurons and, for the first time, human neurons differentiated from pluripotent stem cells. Most notably, tianeptinaline, a class I HDAC-selective analogue of tianeptine, but not tianeptine itself, increased histone acetylation, and enhanced CREB-mediated transcription and the expression of Arc (activity-regulated cytoskeleton-associated protein). Systemic in vivo administration of tianeptinaline to mice confirmed its brain penetration and was found to enhance contextual fear conditioning, a behavioral test of hippocampal-dependent memory. Tianeptinaline and its derivatives provide new pharmacological tools to dissect chromatin-mediated neuroplasticity underlying memory and other epigenetically related processes implicated in health and disease.

Structure activity relationship studies on rhodanines and derived enethiol inhibitors of metallo-β-lactamases

Metallo-β-lactamases (MBLs) enable bacterial resistance to almost all classes of β-lactam antibiotics. We report studies on enethiol containing MBL inhibitors, which were prepared by rhodanine hydrolysis. The enethiols inhibit MBLs from different subclasses. Crystallographic analyses reveal that the enethiol sulphur displaces the di-Zn(II) ion bridging ‘hydrolytic’ water. In some, but not all, cases biophysical analyses provide evidence that rhodanine/enethiol inhibition involves formation of a ternary MBL enethiol rhodanine complex. The results demonstrate how low molecular weight active site Zn(II) chelating compounds can inhibit a range of clinically relevant MBLs and provide additional evidence for the potential of rhodanines to be hydrolysed to potent inhibitors of MBL protein fold and, maybe, other metallo-enzymes, perhaps contributing to the complex biological effects of rhodanines. The results imply that any medicinal chemistry studies employing rhodanines (and related scaffolds) as inhibitors should as a matter of course include testing of their hydrolysis products.

Generation and phenotypic characterization of Pde1a mutant mice.

It has been proposed that a reduction in intracellular calcium causes an increase in intracellular cAMP and PKA activity through stimulation of calcium inhibitable adenylyl cyclase 6 and inhibition of phosphodiesterase 1 (PDE1), the main enzymes generating and degrading cAMP in the distal nephron and collecting duct, thus contributing to the development and progression of autosomal dominant polycystic kidney disease (ADPKD). In zebrafish pde1a depletion aggravates and overexpression ameliorates the cystic phenotype. To study the role of PDE1A in a mammalian system, we used a TALEN pair to Pde1a exon 7, targeting the histidine-aspartic acid dipeptide involved in ligating the active site Zn++ ion to generate two Pde1a null mouse lines. Pde1a mutants had a mild renal cystic disease and a urine concentrating defect (associated with upregulation of PDE4 activity and decreased protein kinase A dependent phosphorylation of aquaporin-2) on a wild-type genetic background and aggravated renal cystic disease on a Pkd2WS25/- background. Pde1a mutants additionally had lower aortic blood pressure and increased left ventricular (LV) ejection fraction, without a change in LV mass index, consistent with the high aortic and low cardiac expression of Pde1a in wild-type mice. These results support an important role of PDE1A in the renal pathogenesis of ADPKD and in the regulation of blood pressure.

Roles of the Active Site Zn(II) and Residues in Substrate Discrimination by Threonyl-tRNA Synthetase: An MD and QM/MM Investigation.

Threonyl-tRNA synthetase (ThrRS) is a Zn(II) containing enzyme that catalyzes the activation of threonine and its subsequent transfer to the cognate tRNA. This process is accomplished with remarkable fidelity, with ThrRS being able to discriminate its cognate substrate from similar analogues such as serine and valine. Molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) methods have been used to elucidate the role of Zn(II) in the aminoacylation mechanism of ThrRS. More specifically, the role of Zn(II) and active site residues in ThrRS's ability to discriminate between its cognate substrate l-threonine and the noncognate l-serine, l-valine, and d-threonine has been examined. The present results suggest that a role of the Zn(II) ion, with its Lewis acidity, is to facilitate deprotonation of the side chain hydroxyl groups of the aminoacyl moieties of cognate Thr-AMP and noncognate Ser-AMP substrates. In their deprotonated forms, these substrates are able to adopt a conformation preferable for aminoacyl transfer from aa-AMP onto the Ado-3'OH of the tRNAThr cosubstrate. Relative to the neutral substrates, when the substrates are first deprotonated with the assistance of the Zn(II) ion, the barrier for the rate-limiting step is decreased significantly by 42.0 and 39.2 kJ mol-1 for l-Thr-AMP and l-Ser-AMP, respectively. An active site arginyl also plays a key role in stabilizing the buildup of negative charge on the substrate's bridging phosphate oxygen during the mechanism. For the enantiomeric substrate analogue d-Thr-AMP, product formation is highly disfavored, and as a result, the reverse reaction has a very low barrier of 16.0 kJ mol-1.

Effect of donor atom identity on metal-binding pharmacophore coordination.

The inhibition and binding of three metal-binding pharmacophores (MBPs), 2-hydroxycyclohepta-2,4,6-trien-1-one (tropolone), 2-mercaptopyridine-N-oxide (1,2-HOPTO), and 2-hydroxycyclohepta-2,4,6-triene-1-thione (thiotropolone) to human carbonic anhydrase II (hCAII) and a mutant protein hCAII L198G were investigated. These MBPs displayed bidentate coordination to the active site Zn(II) metal ion, but the MBPs respond to the mutation of L198G differently, as characterized by inhibition activity assays and X-ray crystallography. The L198G mutation increases the active site volume thereby decreasing the steric pressure exerted on MBPs upon binding, allowing changes in MBP coordination to be observed. When comparing the binding mode of tropolone to thiotropolone or 1,2-HOPTO (O,O versus O,S donor sets), structural modifications of the hCAII active site were shown to have a stronger effect on MBPs with an O,O versus O,S donor set. These findings were corroborated with density functional theory (DFT) calculations of model coordination complexes. These results suggest that the MBP binding geometry is a malleable interaction, particularly for certain ligands, and that the identity of the donor atoms influences the response of the ligand to changes in the protein active site environment. Understanding underlying interactions between a MBP and a metalloenzyme active site may aid in the design and development of potent metalloenzyme inhibitors.

Utility of thermo-alkali-stable γ-CA from polyextremophilic bacterium Aeribacillus pallidus TSHB1 in biomimetic sequestration of CO2 and as a virtual peroxidase.

Aeribacillus pallidus TSHB1 polyextremophilic bacterium produces a γ-carbonic anhydrase (ApCA), which is a homotrimeric biocatalyst with a subunit molecular mass of 32±2kDa. The enzyme is stable in the pH range between 8.0 and 11.0 and thus alkali-stable and moderately thermostable with T1/2 values of 40±1, 15±1, and 8±0.5min at 60, 70, and 80°C, respectively. Activation energy for irreversible inactivation "E d " of carbonic anhydrase is 67.119kJmol-1. The enzyme is stable in the presence of various flue gas contaminants such as SO32-,SO42-, and NO3- and cations Mg2+, Mn2+, Ca2+, and Ba2+. Fluorescence studies in the presence of N-bromosuccinimide and fluorescence quenching using KI and acrylamide revealed the importance of tryptophan residues in maintaining the structural integrity of the enzyme. ApCA is more efficient than the commercially available bovine carbonic anhydrase (BCA) in CO2 sequestration. The enzyme was successfully used in biomineralization of CO2 from flue gas. Replacement of active site Zn2+ with Mn2+ enabled ApCA to function as a peroxidase which exhibited alkali-stability and moderate thermostability like ApCA.

Biophysical and Structural Studies of Chelator Fragment‐Library Candidates as Inhibitors of Metallo‐β‐lactamases

Antibiotic resistance is a serious worldwide health problem. In the 2013 Antibiotic Resistance Threat Report, the CDC estimates that over 2 million people suffer from antibiotic‐resistant infections every year in the U.S. and 23,000 of these individuals die as a result. Metallo‐β‐lactamases are a class of enzymes that catalyze the hydrolysis of almost all β‐lactam antibiotics and, as a result, confer dangerous antibiotic resistance to the bacteria expressing them. Gram‐negative bacteria possessing metallo‐β‐lactamases are of utmost concern because these enzymes also break down carbapenems, a class of antibiotics commonly used as a last resort to treat antibiotic‐resistant bacterial infections. Enterobacteriaceae with carbapenemase activity are becoming increasingly widespread. This is a major problem because these gram‐negative bacteria of the intestinal flora are easily spread between humans and commonly found in clinical settings. Metallo‐β‐lactamase inhibitors are of particular interest because they could potentially restore the efficacy of current generation β‐lactam antibiotics. A library of chelator fragments has been developed to inhibit metallo‐β‐lactamases by taking advantage of the active site Zn2+ ions critical for proper enzymatic function. Our in vitro activity assays have identified several potent candidates from the library for further study. VIM‐2, a variant of Verona integron‐encoded metallo‐β‐lactamase (VIM), was overexpressed and purified for the purpose of studying these chelator fragments. We utilized differential scanning fluorimetry to demonstrate that high concentrations of chelators destabilize VIM‐2 by removing Zn2+ cofactors. Co‐crystallization and crystal soaks of VIM‐2 with several chelators was attempted in order to gain insight into the structure of the protein‐ligand complex but x‐ray diffraction data suggests that the chelators are not present in the crystal structure. Future work will involve binding affinity studies by isothermal titration calorimetry (ITC) and structural studies with advanced crystallographic techniques. Additional studies will be employed to examine the importance of a loop near the active site of all metallo‐β‐lactamases. Overall, these studies will contribute to the development of more effective chelators.Support or Funding InformationThis work was supported by NIH grant R01 GM111926.

Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX

BackgroundHuman carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding. MethodsThe observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry. ResultsThe pKa of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was −24kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01nM and the observed affinity of 2nM. ConclusionsThe intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship. General significanceIt is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations.

Meropenem and chromacef intermediates observed in IMP-25 metallo-β-lactamase-catalyzed hydrolysis.

Metallo-β-lactamases inactivate most β-lactam antibacterials, and much attention has been paid to their catalytic mechanism. One issue of controversy has been whether β-lactam hydrolysis generally proceeds through an anionic intermediate bound to the active-site Zn(II) ions or not. The formation of an intermediate has not been shown conclusively in imipenemase (IMP) enzymes to date. Here, we provide evidence that intermediates are formed during the hydrolysis of meropenem and chromacef catalyzed by the variant IMP-25 and, to a lesser degree, IMP-1.

Variable active site loop conformations accommodate the binding of macrocyclic largazole analogues to HDAC8.

The macrocyclic depsipeptide Largazole is a potent inhibitor of metal-dependent histone deacetylases (HDACs), some of which are drug targets for cancer chemotherapy. Indeed, Largazole partially resembles Romidepsin (FK228), a macrocyclic depsipeptide already approved for clinical use. Each inhibitor contains a pendant side chain thiol that coordinates to the active site Zn(2+) ion, as observed in the X-ray crystal structure of the HDAC8-Largazole complex [Cole, K. E., Dowling, D. P., Boone, M. A., Phillips, A. J., and Christianson, D. W. (2011) J. Am. Chem. Soc. 133, 12474]. Here, we report the X-ray crystal structures of HDAC8 complexed with three synthetic analogues of Largazole in which the depsipeptide ester is replaced with a rigid amide linkage. In two of these analogues, a six-membered pyridine ring is also substituted (with two different orientations) for the five-membered thiazole ring in the macrocycle skeleton. The side chain thiol group of each analogue coordinates to the active site Zn(2+) ion with nearly ideal geometry, thereby preserving the hallmark structural feature of inhibition by Largazole. Surprisingly, in comparison with the binding of Largazole, these analogues trigger alternative conformational changes in loops L1 and L2 flanking the active site. However, despite these structural differences, inhibitory potency is generally comparable to, or just moderately less than, the inhibitory potency of Largazole. Thus, this study reveals important new structure-affinity relationships for the binding of macrocyclic inhibitors to HDAC8.

The role of His-83 of yeast apurinic/apyrimidinic endonuclease Apn1 in catalytic incision of abasic sites in DNA

BackgroundThe apurinic/apyrimidinic (AP) endonuclease Apn1 from Saccharomyces cerevisiae is a key enzyme involved in the base excision repair (BER) at the cleavage stage of abasic sites (AP sites) in DNA. The crystal structure of Apn1 from S. cerevisiae is unresolved. Based on its high amino acid homology to Escherichia coli Endo IV, His-83 is believed to coordinate one of three Zn2+ ions in Apn1's active site similar to His-69 in Endo IV. Substituting His-83 with Ala is proposed to decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn2+ ions involved in enzymatic catalysis. MethodsThe kinetics of recognition, binding, and incision of DNA substrates with the H83A Apn1 mutant was investigated. The stopped-flow method detecting fluorescence intensity changes of 2-aminopurine (2-aPu) was used to monitor the conformational dynamics of DNA at pre-steady-state conditions. ResultsWe found substituting His-83 with Ala influenced catalytic complex formation and further incision of the damaged DNA strand. The H83A Apn1 catalysis depends not only on the location of the mismatch relative to the abasic site in DNA, but also on the nature of damage. ConclusionsWe consider His-83 properly coordinates the active site Zn2+ ion playing a crucial role in catalytic incision stage. Our data prove suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn2+ ions. General significanceOur study provides insights into mechanistic specialty of AP site repair by yeast AP endonuclease Apn1 of Endo IV family, which members are not found in mammals, but are present in many microorganisms. The results will provide useful guidelines for design of new anti-fungal and anti-malarial agents.

Peptides derived from Rhopilema esculentum hydrolysate exhibit angiotensin converting enzyme (ACE) inhibitory and antioxidant abilities.

Jellyfish (Rhopilema esculentum) was hydrolyzed using alcalase, and two peptides with angiotensin-I-converting enzyme (ACE) inhibitory and antioxidant activities were purified by ultrafiltration and consecutive chromatographic methods. The amino acid sequences of the two peptides were identified as VKP (342 Da) and VKCFR (651 Da) by electrospray ionization tandem mass spectrometry. The IC50 values of ACE inhibitory activities of the two peptides were 1.3 μM and 34.5 μM, respectively. Molecular docking results suggested that VKP and VKCFR bind to ACE through coordinating with the active site Zn(II) atom. Free radical scavenging activity and protection against hydrogen peroxide (H2O2)-induced rat cerebral microvascular endothelial cell (RCMEC) injury were used to evaluate the antioxidant activities of the two peptides. As the results clearly showed that the peptides increased the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-px) activities in RCMEC cells), it is proposed that the R. esculentum peptides exert significant antioxidant effects.

In vitro biological evaluation of glyburide as potential inhibitor of collagenases

In tissues with upregulated MMP activity, MMP inhibition remains one of the key strategies. Potential inhibitors of MMPs have been tested for almost 30 years, but none have reached clinical utility due to bioavailability issues and adverse effects. This study utilized the approach of drug repurposing for exploring glyburide as a potential inhibitor against collagenases. In silico molecular docking studies were carried out to probe the interactions of glyburide with the active site Zn. Collagenase enzyme activity measurements and zymography analyses using conditioned medium from lung fibroblasts, rheumatoid synovial fibroblasts, and osteoblasts were carried out to confirm the inhibitory activity. Glyburide binds and interacts with the catalytic Zn residues of the collagenases, as evidenced by in silico molecular docking studies. Fluorescence enzyme activity measurements reveal that glyburide inhibits peptide substrate cleavage by all three collagenases in a dose-dependent manner. Collagen zymography studies validated inhibition of these collagenases by glyburide. These results identify glyburide as a potential inhibitor of collagenases and provide an insight into the mechanism of action of this small molecule. Thus, glyburide may offer additional advantages in diabetics, in controlling MMP activation and collagen degradation and could aid in the treatment of diseases with aberrant MMP activity.

Picosecond-resolved fluorescent probes at functionally distinct tryptophans within a thermophilic alcohol dehydrogenase: relationship of temperature-dependent changes in fluorescence to catalysis.

Two single-tryptophan variants were generated in a thermophilic alcohol dehydrogenase with the goal of correlating temperature-dependent changes in local fluorescence with the previously demonstrated catalytic break at ca. 30 °C (Kohen et al., Nature1999, 399, 496). One tryptophan variant, W87in, resides at the active site within van der Waals contact of bound alcohol substrate; the other variant, W167in, is a remote-site surface reporter located >25 Å from the active site. Picosecond-resolved fluorescence measurements were used to analyze fluorescence lifetimes, time-dependent Stokes shifts, and the extent of collisional quenching at Trp87 and Trp167 as a function of temperature. A subnanosecond fluorescence decay rate constant has been detected for W87in that is ascribed to the proximity of the active site Zn2+ and shows a break in behavior at 30 °C. For the remainder of the reported lifetime measurements, there is no detectable break between 10 and 50 °C, in contrast with previously reported hydrogen/deuterium exchange experiments that revealed a temperature-dependent break analogous to catalysis (Liang et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9556). We conclude that the motions that lead to the rigidification of ht-ADH below 30 °C are likely to be dominated by global processes slower than the picosecond to nanosecond motions measured herein. In the case of collisional quenching of fluorescence by acrylamide, W87in and W167in behave in a similar manner that resembles free tryptophan in water. Stokes shift measurements, by contrast, show distinctive behaviors in which the active-site tryptophan relaxation is highly temperature-dependent, whereas the solvent-exposed tryptophan’s dynamics are temperature-independent. These data are concluded to reflect a significantly constrained environment surrounding the active site Trp87 that both increases the magnitude of the Stokes shift and its temperature-dependence. The results are discussed in the context of spatially distinct differences in enthalpic barriers for protein conformational sampling that may be related to catalysis.

Insights into the mechanism of deubiquitination by JAMM deubiquitinases from cocrystal structures of the enzyme with the substrate and product.

AMSH, a conserved zinc metallo deubiquitinase, controls downregulation and degradation of cell-surface receptors mediated by the endosomal sorting complexes required for transport (ESCRT) machinery. It displays high specificity toward the Lys63-linked polyubiquitin chain, which is used as a signal for ESCRT-mediated endosomal–lysosomal sorting of receptors. Herein, we report the crystal structures of the catalytic domain of AMSH orthologue Sst2 from fission yeast, its ubiquitin (product)-bound form, and its Lys63-linked diubiquitin (substrate)-bound form at 1.45, 1.7, and 2.3 Å, respectively. The structures reveal that the P-side product fragment maintains nearly all the contacts with the enzyme as seen with the P portion (distal ubiquitin) of the Lys63-linked diubiquitin substrate, with additional coordination of the Gly76 carboxylate group of the product with the active-site Zn2+. One of the product-bound structures described herein is the result of an attempt to cocrystallize the diubiquitin substrate bound to an active site mutant presumed to render the enzyme inactive, instead yielding a cocrystal structure of the enzyme bound to the P-side ubiquitin fragment of the substrate (distal ubiquitin). This fragment was generated in situ from the residual activity of the mutant enzyme. In this structure, the catalytic water is seen placed between the active-site Zn2+ and the carboxylate group of Gly76 of ubiquitin, providing what appears to be a snapshot of the active site when the product is about to depart. Comparison of this structure with that of the substrate-bound form suggests the importance of dynamics of a flexible flap near the active site in catalysis. The crystal structure of the Thr319Ile mutant of the catalytic domain of Sst2 provides insight into structural basis of microcephaly capillary malformation syndrome. Isothermal titration calorimetry yields a dissociation constant (KD) of 10.2 ± 0.6 μM for the binding of ubiquitin to the enzyme, a value comparable to the KM of the enzyme catalyzing hydrolysis of the Lys63-linked diubiquitin substrate (∼20 μM). These results, together with the previously reported observation that the intracellular concentration of free ubiquitin (∼20 μM) exceeds that of Lys63-linked polyubiquitin chains, imply that the free, cytosolic form of the enzyme remains inhibited by being tightly bound to free ubiquitin. We propose that when AMSH associates with endosomes, inhibition would be relieved because of ubiquitin binding domains present on its endosomal binding partners that would shift the balance toward better recognition of polyubiquitin chains via the avidity effect.

Identifying functional mimetics of histone deacetylase using isothermal titration calorimetry (LB242)

Inhibition of histone deacetylase (HDAC) is known to induce tumor cell death, making it a promising approach for the treatment of cancer. To determine the energetic contribution of the active‐site Zn2+ to HDAC interaction with inhibitors as superoylanilide hydroxamic acid (SAHA), we have explored Zn2+ chelators as functional mimetics of HDAC as an alternative to HDAC enzyme. Our goal was to first identify Zn2+ chelators that are capable of forming tight 1:1 (Zn2+:chelator) binary complex and then to determine the energetics of inhibitor binding to the binary complex. One chelator, N‐(2‐(1‐methylimidazolyl)methyL)iminodiacetic acid (DA2Im), was synthesized and three others, Nitrilotriacetic acid (NTA), Tris(2‐pyridylmethyl)amine (TPA) and Tris(2‐aminoethyl)amine (TREN), were purchased from commercial sources. Their Zn2+ binding stoichiometry and thermodynamic parameters were determined using isothermal titration calorimetry (ITC) in either a NEM buffer (pH 6.8) or a methanol:buffer mixture (pH 6.8) with the methanol added to increase the solubility of the less polar chelators. DA2Im and NTA were found to bind with a stoichiometry equivalent to three chelators for every two Zn2+ and an equilibrium constant of (1.2±0.3)×108 and (2.1±0.6)×107 M‐1, respectively, in the buffer without methanol. Conversely, TREN and TPA were found to bind Zn2+ with the desired 1:1 stoichiometry and an equilibrium constant of (1.5±0.1)×106 and >107 M‐1, respectively, in the methanol:buffer mixture. We conclude that TPA and TREN are promising candidates as HDAC functional mimetics for studying Zn2+ inhibitor interactions. ITC experiments are currently underway to use the Zn2+:chelator binary complexes to obtain the energetic contribution of active site Zn2+ to HDAC interaction with inhibitors.