A unique aromatic cluster near the active site of H. pylori CPA is essential for catalytic function

Arginase is a bimetallic enzyme that utilizes mainly Mn2+ or Co2+ for catalytic function. In human homolog, the substitution of Mn2+ with Co2+ significantly reduces the Km value without affecting the kcat. However, in the Helicobacter pylori counterpart (important for pathogenesis), the kcat increases nearly 4-fold with Co2+ ions both in the recombinant holoenzyme and arginase isolated from H. pylori grown with Co2+ or Mn2+. This suggests that the active site of arginase in the two homologs is modulated differently by these two metal ions. To investigate the underlying mechanism for metal-induced difference in catalytic activity in the H. pylori enzyme, we used biochemical, biophysical and microsecond molecular dynamics simulations studies. The study shows that the difference in binding affinity of Co2+ and Mn2+ ions with the protein is linked to a different positioning of a loop (–122HTAYDSDSKHIHG134–) that contains a conserved catalytic His133. Consequently, the proximity of His133 and conserved Glu281 is varied. We found that the Glu281–His133 interaction is crucial for catalytic function and was previously unexplored in other homologs. We suggest that the proximity difference between these two residues in the Co2+- and Mn2+-proteins alters the proportion of protonated His133 via variation in its pKa. This affects the efficiency of proton transfer — an essential step of l-arginine hydrolysis reaction catalyzed by arginase and thus activity. Unlike in human arginase, the flexibility of the above segment observed in H. pylori homolog suggests that this region in the H. pylori enzyme may be explored to design its specific inhibitors.

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Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/156.1.21 ◽

2000 ◽

Vol 156 (1) ◽

pp. 21-29 ◽

Cited By ~ 1

Author(s):

David R H Evans ◽

Brian A Hemmings

Keyword(s):

Protein Interactions ◽

Active Site ◽

Protein Function ◽

Mutational Analysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Active Site Residues ◽

Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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In Vivo Consequences of Putative Active Site Mutations in Yeast DNA Polymerases α, ε, δ, and ζ

Genetics ◽

10.1093/genetics/159.1.47 ◽

2001 ◽

Vol 159 (1) ◽

pp. 47-64 ◽

Cited By ~ 1

Author(s):

Youri I Pavlov ◽

Polina V Shcherbakova ◽

Thomas A Kunkel

Keyword(s):

Active Site ◽

Dna Polymerases ◽

Base Substitution ◽

Loss Of Function ◽

Chromosomal Dna ◽

Strong Base ◽

Dna Mismatch ◽

Catalytic Function ◽

Base Substitutions

Abstract Several amino acids in the active site of family A DNA polymerases contribute to accurate DNA synthesis. For two of these residues, family B DNA polymerases have conserved tyrosine residues in regions II and III that are suggested to have similar functions. Here we replaced each tyrosine with alanine in the catalytic subunits of yeast DNA polymerases α, δ, ε, and ζ and examined the consequences in vivo. Strains with the tyrosine substitution in the conserved SL/MYPS/N motif in region II in Polδ or Polε are inviable. Strains with same substitution in Rev3, the catalytic subunit of Polζ, are nearly UV immutable, suggesting severe loss of function. A strain with this substitution in Polα (pol1-Y869A) is viable, but it exhibits slow growth, sensitivity to hydroxyurea, and a spontaneous mutator phenotype for frameshifts and base substitutions. The pol1-Y869A/pol1-Y869A diploid exhibits aberrant growth. Thus, this tyrosine is critical for the function of all four eukaryotic family B DNA polymerases. Strains with a tyrosine substitution in the conserved NS/VxYG motif in region III in Polα, -δ, or -ε are viable and a strain with the homologous substitution in Rev3 is UV mutable. The Polα mutant has no obvious phenotype. The Polε (pol2-Y831A) mutant is slightly sensitive to hydroxyurea and is a semidominant mutator for spontaneous base substitutions and frameshifts. The Polδ mutant (pol3-Y708A) grows slowly, is sensitive to hydroxyurea and methyl methanesulfonate, and is a strong base substitution and frameshift mutator. The pol3-Y708A/pol3-Y708A diploid grows slowly and aberrantly. Mutation rates in the Polα, -δ, and -ε mutant strains are increased in a locus-specific manner by inactivation of PMS1-dependent DNA mismatch repair, suggesting that the mutator effects are due to reduced fidelity of chromosomal DNA replication. This could result directly from relaxed base selectivity of the mutant polymerases due to the amino acid changes in the polymerase active site. In addition, the alanine substitutions may impair catalytic function to allow a different polymerase to compete at the replication fork. This is supported by the observation that the pol3-Y708A mutation is recessive and its mutator effect is partially suppressed by disruption of the REV3 gene.

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Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme

RNA ◽

10.1261/rna.067579.118 ◽

2018 ◽

Vol 24 (11) ◽

pp. 1542-1554 ◽

Cited By ~ 2

Author(s):

Neil A. White ◽

Minako Sumita ◽

Victor E. Marquez ◽

Charles G. Hoogstraten

Keyword(s):

Active Site ◽

Conformational Dynamics ◽

Catalytic Function

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Design and Characterisation of Inhibitory Peptides against Bleg1_2478, an Evolutionary Divergent B3 Metallo-β-lactamase

Molecules ◽

10.3390/molecules25245797 ◽

2020 ◽

Vol 25 (24) ◽

pp. 5797

Author(s):

Gayathri Selvaraju ◽

Thean Chor Leow ◽

Abu Bakar Salleh ◽

Yahaya M. Normi

Keyword(s):

Active Site ◽

Protein Complexes ◽

Hypothetical Protein ◽

Titration Calorimetry ◽

Binding Properties ◽

Vitro Assay ◽

In Silico Docking ◽

Inhibitory Peptides ◽

Catalytic Function

Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-β-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide–protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.

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Structure analysis of endosialidase NF at 0.98 Å resolution

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444909048720 ◽

2010 ◽

Vol 66 (2) ◽

pp. 176-180 ◽

Cited By ~ 8

Author(s):

Eike C. Schulz ◽

Piotr Neumann ◽

Rita Gerardy-Schahn ◽

George M. Sheldrick ◽

Ralf Ficner

Keyword(s):

Sialic Acid ◽

Structure Analysis ◽

Active Site ◽

Polysialic Acid ◽

Crystal Form ◽

Atomic Resolution ◽

Active Site Residue ◽

Catalytic Function ◽

Multiple Conformations ◽

A Minor

Endosialidase NF (endoNF) is a bacteriophage-derived endosialidase that specifically degrades α-2,8-linked polysialic acid. The structure of a new crystal form of endoNF in complex with sialic acid has been refined at 0.98 Å resolution. The 210 kDa homotrimeric multi-domain enzyme displays outstanding stability and resistance to SDS. Even at atomic resolution, only a minor fraction of side chains possess alternative conformations. However, multiple conformations of an active-site residue imply that it has an important catalytic function in the cleavage mechanism of polysialic acid.

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Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function

Biochemical Journal ◽

10.1042/bj2720479 ◽

1990 ◽

Vol 272 (2) ◽

pp. 479-484 ◽

Cited By ~ 17

Author(s):

C Andersson ◽

R Morgenstern

Keyword(s):

Chemical Modification ◽

Rat Liver ◽

Active Site ◽

Glutathione Transferase ◽

Amino Acid Residues ◽

Active Site Residues ◽

Histidine Residues ◽

Catalytic Function ◽

Low Efficiency ◽

Microsomal Glutathione

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5′-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.

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Effect of mutation of lysine-128 of the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans

Biochemical Journal ◽

10.1042/bj3360387 ◽

1998 ◽

Vol 336 (2) ◽

pp. 387-393 ◽

Cited By ~ 9

Author(s):

Graeme BAINBRIDGE ◽

P. John ANRALOJC ◽

Pippa J. MADGWICK ◽

Jim E. PITTS ◽

Martin A. J. PARRY

Keyword(s):

Aspartic Acid ◽

Active Site ◽

Large Subunit ◽

Ribulose Bisphosphate Carboxylase ◽

Bisphosphate Carboxylase ◽

Catalytic Function ◽

Loop Dynamics ◽

Ribulose Bisphosphate Carboxylase Oxygenase ◽

Genes Encoding ◽

Specificity Factor

The contribution of lysine-128 within the active site of Anacystis nidulansd-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was investigated by the characterization of mutants in which lysine-128 was replaced with arginine, glycine, glutamine, histidine or aspartic acid. Mutated genes encoding the Rubisco large subunit were expressed in Escherichia coliand the resultant polypeptides assembled into active complexes. All of the mutant enzymes had a lower affinity for ribulose 1,5-bisphosphate (RuBP) and lower rates of carboxylation. Substitution of lysine-128 with glutamine, histidine or aspartic acid decreased the specificity factor and led to the production of an additional monophosphate reaction product. We show that this product results from the loss of the phosphate from C-1 of RuBP, most probably by β-elimination from the 2,3-enediolate derivative of RuBP. The results confirm that lysine-128 is important in determining the position of the essential ε-amino group of lysine-334 within the active site and in loop dynamics. This further demonstrates that residues remote from the active site can be manipulated to modify catalytic function.

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Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

eLife ◽

10.7554/elife.07574 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 78

Author(s):

Daniel A Keedy ◽

Lillian R Kenner ◽

Matthew Warkentin ◽

Rahel A Woldeyes ◽

Jesse B Hopkins ◽

...

Keyword(s):

Active Site ◽

Protein Function ◽

Conformational Ensemble ◽

Conformational Heterogeneity ◽

Time Resolved ◽

Protein Motions ◽

Catalytic Function ◽

Temperature Dependences ◽

Site Network ◽

Response To Temperature

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.

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Modulating Enzyme Function via Dynamic Allostery within Biliverdin Reductase B

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.691208 ◽

2021 ◽

Vol 8 ◽

Author(s):

Jasmina S. Redzic ◽

Michael R. Duff ◽

Ashley Blue ◽

Todd M. Pitts ◽

Pratul Agarwal ◽

...

Keyword(s):

Active Site ◽

Redox Regulation ◽

Nmr Relaxation ◽

Functional Coupling ◽

Global Dynamics ◽

Biliverdin Reductase ◽

Coenzyme Binding ◽

Catalytic Function ◽

Order Of Magnitude ◽

And Function

The biliverdin reductase B (BLVRB) class of enzymes catalyze the NADPH-dependent reduction of multiple flavin substrates and are emerging as critical players in cellular redox regulation. However, the role of dynamics and allostery have not been addressed, prompting studies here that have revealed a position 15 Å away from the active site within human BLVRB (T164) that is inherently dynamic and can be mutated to control global micro-millisecond motions and function. By comparing the inherent dynamics through nuclear magnetic resonance (NMR) relaxation approaches of evolutionarily distinct BLVRB homologues and by applying our previously developed Relaxation And Single Site Multiple Mutations (RASSMM) approach that monitors both the functional and dynamic effects of multiple mutations to the single T164 site, we have discovered that the most dramatic mutagenic effects coincide with evolutionary changes and these modulate coenzyme binding. Thus, evolutionarily changing sites distal to the active site serve as dynamic “dials” to globally modulate motions and function. Despite the distal dynamic and functional coupling modulated by this site, micro-millisecond motions span an order of magnitude in their apparent kinetic rates of motions. Thus, global dynamics within BLVRB are a collection of partially coupled motions tied to catalytic function.

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