New Insight into the Catalytic and Inhibition Mechanism of the Human Acyl Protein Thioesterase

Wednesday, March 2, 2016 demonstrate that the active site geometry is preserved. We quantified the changes in proton delocalization equilibrium with UV-vis spectroscopy, 13CNMR, and 1H-NMR. In parallel, we also measured the electrostatic interaction between the enzyme and a carbonyl group on an inhibitor where charge separation occurs during catalysis with vibrational Stark effect spectroscopy. The correlations between the extent of proton delocalization, the strength of electric field, and the catalytic efficiency will be discussed to address the contributions of proton delocalization and electrostatic stabilization in this enzyme’s catalytic mechanism. 2696-Pos Board B73 Vibrational Stark Effects for Diverse Carbonyl Probes Applied to the ReInterpretation of IR and Raman Data in Terms of Electric Fields at Enzyme Active Sites Samuel H. Schneider, Steven G. Boxer. Department of Chemistry, Stanford University, Stanford, CA, USA. Carbonyl groups are ubiquitious in biology and found in many different forms including ketones, oxoesters (e.g. lipids, acyl-intermediates), thioesters (e.g. acetyl-CoA, fatty acid biosynthesis), and amides (e.g. DNA, RNA, proteins). Previous work in our lab has utilized the vibrational Stark effect (VSE) of ketones on non-natural amino acids [1] and substrate analogs [2] to quantify the electric field within several proteins, based on the vibration’s linear Stark tuning rate. In order to further probe diverse biophysical systems and environments, we have measured the sensitivity of a functionally-relevant collection of carbonyl-containing compounds by vibrational Stark spectroscopy (VSS) to obtain the Stark tuning rate, and further calibrated them to an absolute electric field utilizing vibrational solvatochromism and MD simulations. The VSE provides a quantitative framework for interpreting observed frequency shifts in biophysical systems, providing physical insights into binding and catalysis. As such, this calibration to an electric field provides a unifying model with which to re-examine a substantial body of literature on highly unusual IR and Raman data of inhibitors at the enzyme active site, which have been previously attributed to substrate polarization and bond distortion [e.g. 3]. The generalizability of this interpretation to many enzymatic systems further suggests the importance of enzyme electrostatics in modulating function and may have significant applications in protein design and engineering. References 1. Fried, S.D.; Bagchi, S.; Boxer, S.G., J. Am. Chem. Soc., 2013, 135, 1118111192. 2. Fried, S.D.; Bagchi, S.; Boxer, S.G., Science, 2014, 236, 1510-1514. 3. Carey, P.R., Chem. Rev., 2006, 106, 3043-3054. 2697-Pos Board B74 Computational Study on the Catalytic Effect of the Magnesium Ions in the Mechanism of DNA Polymerases Ricardo A. Matute, Arieh Warshel. Department of Chemistry, University of Southern California, Los Angeles, CA, USA. The DNA replication is catalyzed by DNA polymerases upon the nucleophile activation via proton transfer on the corresponding nucleotide of the growing template strand in order to trigger the nucleophilic attack on the incoming nucleotide and release of pyrophosphate as leaving group. In the present work, a computational study on the enzyme catalysis mechanism of the DNA polymerase b was carried out in order to assess changes in the energetics when acidic residues in the active site are mutated. The electrostatic environment in the active site is indeed strongly dependent on the catalytic effects of the magnesium ions for both native and mutant enzyme. 2698-Pos Board B75 The Effect of Magnesium Ion Concentration on the Nucleotide Specificity and Fidelity of HIV-1 Reverse Transcriptase Shanzhong Gong, Kenneth Johnson. Molecular Bioscience, The University of Texas at Austin, Austin, TX, USA. Like other DNA polymerases there are two Mg2þ bound to the polymerase site of HIV-1 reverse transcriptase (HIVRT) and they are thought to facilitate the incorporation of normal nucleotides through a classical two metal ion mechanism. However, the effect of Mg2þ on the fidelity of HIVRT and role of each Mg2þ on the steps governing nucleotide incorporation are unknown and although most in vitro measurements have been performed using 6 -10 mM Mg2þ, it is reported that the physiological concentration of free Mg2þ may be as low as 0.25 mM. In this study, we investigated the effects of Mg2þ on nucleotide specificity by defining the kinetic parameters governing each step involved in the normal nucleotide in the incorporation pathway; namely, ground-state binding, substrate-induced conformational change, substrate release, chemistry and pyrophosphate release. One Mg2þ is chelated by the

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nucleotide, which binds to the enzyme as a Mg-dNTP2- complex, while the second Mg2þ binds in a separate step. Surprisingly, Mg2þ concentrations ranging from 0.25 to 10 mM did not affect the kinetics of nucleotide binding or the rate of the conformational change. Rather, the main effect of free Mg2þ is on the rate of the chemistry step. These results demonstrate that the second Mg2þ binds after the conformational change and with an apparent Kd of 4.7 mM. We also measured the fidelity of HIV-1 RT under various concentrations of Mg2þ and show that Mg2þ does not significantly affect the fidelity at concentrations R 0.25 mM. We also examined the effect of Mg2þ concentration on the kinetics of DNA binding. Overall, the results from our study provide valuable information regarding magnesium concentration and nucleotide specificity. 2699-Pos Board B76 The Role of Phosphorylation and Acetylation of TFAM in DNA Binding Regulation using Single-Molecule Manipulation and Fluorescence Microscopy Maryam Hashemi Shabestari1, Graeme A. King1, Wouter H. Roos1, Carolyn K. Suzuki2, Gijs J.L. Wuite1. 1 Department of Physics and Astronomy and LaserLaB, VU University Amsterdam, Amsterdam, Netherlands, 2Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA. Mitochondrial transcription factor A (TFAM) is a multifunctional protein, which orchestrates mitochondrial DNA compaction, transcription and replication. While post-translational modifications, such as TFAM phosphorylation and acetylation are thought to regulate these processes, the mechanism by which this regulation occurs is poorly understood. Using a combination of single-molecule manipulation and fluorescence microscopy, we investigate the effect of TFAM phosphorylation and acetylation on DNA binding affinity. By fitting force-extension curves of TFAM-bound DNA to the Worm-Like Chain model, we determine how the persistence length of DNA changes with increasing protein concentration. In this way, we determine the binding affinity of TFAM to DNA as well as the extent of TFAM induced DNA compaction. We demonstrate that phosphorylation and acetylation of TFAM do not alter its ability to compact DNA, but significantly lower the binding affinity to DNA. Furthermore, by visualizing fluorescently-labelled TFAM unbinding from DNA, we reveal an increase in the unbinding rate of TFAM from DNA upon phosphorylation. This indicates that the reduced binding affinity of TFAM to DNA when phosphorylated is at least partially due to the higher off-rate of phosphorylated TFAM. Conversely, the unbinding rate of TFAM from DNA remains unaffected by acetylation. Therefore, we relate the lower binding affinity of acetylated TFAM to a decrease in the on-rate of the protein. These findings indicate that phosphorylation and acetylation can regulate TFAM function and may lead to a deeper understanding of the in vivo variations of TFAM coating on DNA and its exact biological function. 2700-Pos Board B77 New Insight into the Catalytic and Inhibition Mechanism of the Human Acyl Protein Thioesterase Martina Audagnotto, Sylvia Ho, Patrick Sandoz, Nicole Andenmatten, Gisou van der Goot, Matteo Dal Peraro. EPFL, Lausanne, Switzerland. Post-translational modifications play a crucial role in regulating the function of many biological molecules. Lipid modifications, in particular, via prenylation, acylation, myristoylation and palmitoylation help to direct membrane localization, protein-protein interactions, cell signaling, subcellular trafficking and vesicle transport. Among them, palmitoylation is the only reversible process. This modification consists in the addition of a palmitic acid forming a thioester bond with cysteine residues of both soluble and membrane proteins. The thioester linkage is hydrolyzed by acyl protein thioesterase enzymes (APTs), which belong to the a/b hydrolase family of serine hydrolases. The first soluble thioesterase to be characterized was the human APT1, which catalyzes depalmitoylation of signaling regulators like Ga and H/N-Ras. This structure revealed a non-symmetric homodimeric organization where the catalytic pocket is occluded by the dimer interface. The human APT1 is itself palmitoylated in position 2 and acts as substrate for itself. Recently, we have solved new X-ray structures for the wild-type and the catalytic inactive form (S119A) of human APT1, which reveal (i) a dimeric interface suggesting a novel mode of interaction with the biological membrane, and (ii) a previously unseen electron density adjacent to the catalytic pocket that allowed to study the enzyme-substrate adduct via molecular simulation. Moreover, this new evidence suggested the existence of a druggable pocket that can favorably accommodate a variety of small molecule compounds as observed

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Wednesday, March 2, 2016

from a molecular docking campaign on several libraries. We are currently testing in vitro the most promising compounds from this in silico screening to identify high affinity hits able to inhibit human APT1. 2701-Pos Board B78 Structural and Biochemical Investigations on the Catalytic Mechanism of Pyridoxal Kinase (PdxK) from Salmonella Typhimurium and its Interactions with PLP-Dependent Enzymes G. Deka1, J.F. Benazir1, J.N. Kalyani2, H.S. Savithri2, M.R.N. Murthy1. 1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India, 2 Biochemistry Department, Indian Institute of Science, Bangalore, India. Pyridoxal kinase (PdxK), a member of ribokinase superfamily of enzymes, is involved in pyridoxal 5’ phosphate (PLP) synthesis by a salvage pathway. PdxK ensures availability of PLP to a large number of enzymes involved in amino acid and sugar metabolism that use PLP as a cofactor and is regarded as a potential drug target. We have determined the crystal structure of PdxK from Salmonella typhimurium (stPLK) in its unliganded form as well ˚ , 1.9A ˚ and 2.5A ˚ resas in complex with Mg ATP and Mg ATP-PLP at 2.6A olutions, respectively. The protomeric structure of the dimeric enzyme consisting of three layered aba structure is similar to those of other ribokinase family proteins. A segment of residues 134-142 constituting a flexible loop undergoes a large conformational change from an open form to a closed form upon ligand binding and guards the active site from solvent exposure and prevents premature hydrolysis of ATP. During catalysis, the substrate ˚ to interact with the g phosphate of ATP bound PL moves by a distance of ~6A near an anion hole constituted by residues 234-237 (GTGD). As reported for the E. coli enzyme, kinetic studies show that stPLK has higher activity in the presence of Mg2þ when compared to other divalent metal ions. High concentration of PLP was found to inhibit stPLK and the structure of crystals obtained in presence of excess PLP reveals that PLP is covalently attached as an internal aldimine to Lys233. Surface Plasmon resonance (SPR ) and ELISA studies show that stPLK specifically interacts with diaminopropionate ammonia lyase, a fold type II PLP dependent enzyme, suggesting probable direct transfer of the product PLP from PdxK to the apo form of PLPdependent enzymes. 2702-Pos Board B79 Molecular Basis for Cohesin Acetylation by Establishment of Sister Chromatid Cohesion N-Acetyltransferase (ESCO1) Yadilette Rivera-Colon, Andrew Maguire, Glen P. Liszczak, Ronen Marmorstein. Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA. Cell division in both prokaryotes and eukaryotes relies on the accurate segregation of chromosomes. The Structural Maintenance of Chromosomes (SMC)heterodimeric ATPase protein complex,cohesin, plays a key role in this process as it is responsible for cohesion between sister chromatids and is a major constituent of interphase and mitotic chromosomes. The functions of cohesin are modulated by phosphorylation, acetylation, ATP hydrolysis, and site-specific proteolysis of its SMC1 and/or SMC3 subunits. Establishment of sister chromatid cohesion is mediated by acetylation of the cohesin subunit SMC3 by the lysine acetyltransferase ESCO1. ESCO2 is a paralogous human cohesin acetyltransferase and genetic defects that reduce ESCO2 activity lead to Roberts Syndrome, a childhood autosomal recessive disorder that manifests by mental retardation, craniofacial abnormalities and limb reduction. We have determined the X-ray crystal structure of ESCO1 and have carried out structure-based mutagenesis, biochemical and enzymatic studies. Together, these studies provide novel insights into SMC3-specific acetylation by ESCO1 and rationalize the functional consequence of ESCO2 mutations correlated with Roberts Syndrome. 2703-Pos Board B80 Origins of Catalytic Specificity in Bacterial Oligosaccharyltransferase Brittany R. Morgan, Francesca Massi. Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA. Modification of polypeptides through asparagine (N)-linked glycosylation is involved in a broad range of biological functions. N-linked glycosylation is catalyzed by oligosaccharyltransferase (OST), an endoplasmic reticulum membrane protein, which promotes the formation of an N-glycosidic linkage between the acceptor asparagine and an oligosaccharide donor. Broad polypeptide substrate specificity is characteristic of N-linked glycosylation, resulting from a short consensus sequence: N-X-S/T (where X is any amino acid except P). However, experimental evidence has shown that the N-glycosylation efficiency is affected by the sequence within this consensus motif.

We investigated the catalytic specificity of Campylobacter lari OST using molecular dynamics simulations. In OST, a large external loop (EL5) pins the substrate in the binding pocket located between the transmembrane and periplasmic domains while two acidic catalytic residues, E319 and D56 in C. lari, are in position to form hydrogen bonds with the acceptor asparagine and prime it for nucleophilic attack on the oligosaccharide donor. To explore how different substrates affect the efficiency of N-linked glycosylation, four substrates of differing N-glycosylation efficiencies were examined: the optimal consensus sequence (NAT) and three sub-optimal variants (NAS, NFS, and NWS). Our simulations of OST in complex with the optimal substrate (NAT) show a conformational change of EL5 and of the periplasmic domain is necessary to promote optimal hydrogen bond formation between the acceptor asparagine and E319/D56 by restricting side chain motion in the catalytic pocket. In addition, we found that binding of the other three substrates (NAS, NFS, and NWS) affects the structure and dynamics of OST. These changes lower the probability of forming hydrogen bonds with the acceptor asparagine, essential for catalysis, and accelerate substrate release for NFS, providing two mechanisms for modulating the glycosylation efficiency of OST for various consensus sequence substrates. 2704-Pos Board B81 Molecular Mechanism of the Catalytic Reaction of no Reductase Revealed by Novel Time-Resolved Visible/IR Absorption Spectrometers with Microfluidic Device Tetsunari Kimura1,2, Hanae Takeda1,3, Shoko Ishii1,3, Takehiko Tosha1, Yoshitsugu Shiro1,3, Minoru Kubo1,4. 1 SPring-8 Ctr., RIKEN, Sayo, Japan, 2Grad. Sch. Sci., Kobe Univ., Kobe, Japan, 3Grad. Sch. Life Sci., Univ. of Hyogo, Kamigori, Japan, 4Presto, JST, Kawaguchi, Japan. Time-resolved (TR) spectroscopy plays convincing roles in clarifying the molecular mechanism of biological reactions in the atomic and electronic level. Most of the biological reactions can be triggered by the sudden changes in buffer conditions, but the time-resolution of the conventional solution-mixing technique is limited to several milliseconds and the sample consumption is enormous, resulting in the limited applications of TR spectroscopy. Here, to investigate the enzymatic reaction of a low-yield membrane protein with microsecond-resolution, novel flow-flash TR-visible/IR spectrometers were developed. Time-resolution of microseconds was achieved using cagedcompounds, which release substrates upon laser flash. Combinational use of a micro-channel flow-cell and a nano-liter step-pulse syringe-pump synchronized with the microscopic laser flashes realized the spectral accumulation with the minimal sample consumption. The developed system was applied to nitric-oxide reductase (NOR), a membrane enzyme that catalyzes NO reduction (2NO þ 2Hþ þ 2e- -> N2O þ H2O) in the bacterium denitrification process. Although our X-ray crystallographic analysis has revealed the atomic structure of catalytic center consisting of heme b3 and non-heme FeB, the molecular mechanism of NO reduction is still controversial. This is due to the difficulties in direct observation of the transient NO-bound form, whose lifetime is shorter than 1 ms. Our newly developed TR-visible absorption spectrometer, which probed the electronic state of heme b3, revealed that NO bound to heme b3 within 4 ms and was reduced with a time constant of 100 ms. TR-IR measurement at 10 ms showed that another NO molecule bound to FeB. These TR measurements revealed that each iron in the active center binds different NO molecule in the early stage of the reaction and the subsequent N-N bond formation occurs in the intramolecular manner. 2705-Pos Board B82 Droplet-Based Microfluidics for Measuring Enzymatic Activities: Application to L-Asparaginase used in Antileukemic Therapy Manfred W. Konrad1, Christos S. Karamitros1, Joanan Lopez Morales1, Jean-Christophe Baret2. 1 Enzyme Biochemistry, Max Planck Institute, Goettingen, Germany, 2Soft MicroSystems, CRPP, CNRS, University of Bordeaux, Bordeaux, France. Our work aims to ameliorate catalytic properties of L-asparaginase (L-ASNase) which is a protein drug used in antileukemic therapy. Bacterial L-ASNases are FDA-approved therapeutic enzymes for use in the treatment of various blood cancers to deplete serum L-Asn. Their therapeutic efficacy is based on the fact that several hematological malignancies, in particular Acute Lymphoblastic Leukemia (ALL), depend for growth on the extracellular supply of L-Asn. To avoid various side reactions inherent to the bacterial enzymes, it would be beneficial to substitute them with human L-ASNases. In order to find variants of improved specific activities in mutated enzyme libraries, we developed a droplet-based microfluidic platform for high throughput and miniaturization of kinetic assays that can be performed not only on purified