Arginine 343 and 350 Are Two Active Site Residues Involved in Substrate Binding by Human Type I D-myo-Inositol 1,4,5-Trisphosphate 5-Phosphatase

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

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Substrate Binding and Catalysis by Ubiquitin C-Terminal Hydrolases: Identification of Two Active Site Residues†

Biochemistry ◽

10.1021/bi960099f ◽

1996 ◽

Vol 35 (21) ◽

pp. 6735-6744 ◽

Cited By ~ 146

Author(s):

Christopher N. Larsen ◽

Joanne S. Price ◽

Keith D. Wilkinson

Keyword(s):

Active Site ◽

Substrate Binding ◽

Active Site Residues

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Evidence that Asn542 of neprilysin (EC 3.4.24.11) is involved in binding of the P2′ residue of substrates and inhibitors

Biochemical Journal ◽

10.1042/bj3110623 ◽

1995 ◽

Vol 311 (2) ◽

pp. 623-627 ◽

Cited By ~ 24

Author(s):

N Dion ◽

H Le Moual ◽

M C Fournié-Zaluski ◽

B P Roques ◽

P Crine ◽

...

Keyword(s):

Active Site ◽

Substrate Binding ◽

Biologically Active ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Active Site Residues ◽

Hydrophobic Cluster Analysis ◽

Active Peptides ◽

Ic50 Values

Neprilysin (EC 3.4.24.11) is a Zn2+ metallopeptidase involved in the degradation of biologically active peptides, e.g. enkephalins and atrial natriuretic peptide. The substrate specificity and catalytic activity of neprilysin resemble those of thermolysin, a crystallized bacterial Zn2+ metalloprotease. Despite little overall homology between the primary structures of thermolysin and neprilysin, many of the amino acid residues involved in catalysis, as well as Zn2+ and substrate binding, are highly conserved. Most of the active-site residues of neprilysin have their homologues in thermolysin and have been characterized by site-directed mutagenesis. Furthermore, hydrophobic cluster analysis has revealed some other analogies between the neprilysin and thermolysin sequences [Benchetrit, Bissery, Mornon, Devault, Crine and Roques (1988) Biochemistry 27, 592-596]. According to this analysis the role of Asn542 in the neprilysin active site is analogous to that of Asn112 of thermolysin, which is to bind the substrate. Site-directed mutagenesis was used to change Asn542 to Gly or Gln residues. The effect of these mutations on substrate catalysis and inhibitor binding was examined with a series of thiorphan-like compounds containing various degrees of methylation at the P2′ residue. For both mutated enzymes, determination of kinetic parameters with [D-Ala2,Leu5]enkephalin as substrate showed that the large decrease in activity was attributable to an increase in Km (14-16-fold) whereas kcat values were only slightly affected (2-3-fold decrease). This is in agreement with Asn542 being involved in substrate binding rather than directly in catalysis. Finally, the IC50 values for thiorphan and substituted thiorphans strongly suggest that Asn542 of neprilysin binds the substrate on the amino side of the P2′ residue by formation of a unique hydrogen bond.

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A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525783113 ◽

2016 ◽

Vol 113 (8) ◽

pp. 2068-2073 ◽

Cited By ~ 32

Author(s):

Erik W. Debler ◽

Kanishk Jain ◽

Rebeccah A. Warmack ◽

You Feng ◽

Steven G. Clarke ◽

...

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Structural Basis ◽

Titration Calorimetry ◽

Type I ◽

Histone H4 ◽

Active Site Residues ◽

Protein Arginine Methyltransferase ◽

Arginine Methyltransferase ◽

Product Specificity

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

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Crystallographic Comparison of Manganese- and Iron-Dependent Homoprotocatechuate 2,3-Dioxygenases

Journal of Bacteriology ◽

10.1128/jb.186.7.1945-1958.2004 ◽

2004 ◽

Vol 186 (7) ◽

pp. 1945-1958 ◽

Cited By ~ 114

Author(s):

Matthew W. Vetting ◽

Lawrence P. Wackett ◽

Lawrence Que ◽

John D. Lipscomb ◽

Douglas H. Ohlendorf

Keyword(s):

Active Site ◽

Metal Ion ◽

Ion Selectivity ◽

Axial Ligand ◽

Activation Process ◽

Type I ◽

Active Site Residues ◽

Metal Ion Selectivity ◽

Mean Square Difference ◽

Extradiol Dioxygenases

ABSTRACT The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn2+ and Fe2+, respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four βαβββ modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214NE1 and E267OE1; one axial ligand, H155NE1; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 Å), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An “open” coordination site trans to E267 is the likely binding site for O2. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O2 during catalysis are identified and discussed in the context of current mechanistic hypotheses.

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Structural basis for the differential regulatory roles of the PDZ domain in C-terminal processing proteases

10.1101/621037 ◽

2019 ◽

Author(s):

Chuang-Kai Chueh ◽

Nilanjan Som ◽

Lu-Chu Ke ◽

Meng-Ru Ho ◽

Manjula Reddy ◽

...

Keyword(s):

Functional Analysis ◽

Active Site ◽

Resting State ◽

Substrate Binding ◽

Pdz Domain ◽

Structural Basis ◽

Active Site Residues ◽

Structural Remodeling ◽

Hydrophobic Substrate ◽

Specific Protease

AbstractCarboxyl (C)-terminal processing proteases (CTPs) participate in protective and regulatory proteolysis in bacteria. The PDZ domain is central to the activity of CTPs but plays inherently different regulatory roles. For example, the PDZ domain inhibits the activity of the signaling protease CtpB by blocking the active site but is required for the activation of Prc (or Tsp), a tail-specific protease that degrades the ssrA-tagged proteins. Here, by structural and functional analysis we show that in the unliganded resting state of Prc, the PDZ domain is docked inside the bowl-shaped scaffold without contacting the active site, which is kept in a default misaligned conformation. In Prc, a hydrophobic substrate sensor distinct from CtpB engages substrate binding to the PDZ domain and triggers a structural remodeling to align the active site residues. Therefore, this work reveals the structural basis for understanding the contrasting roles of the PDZ domain in the regulation of CTPs.

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Structural Basis for the Differential Regulatory Roles of the PDZ Domain in C-Terminal Processing Proteases

mBio ◽

10.1128/mbio.01129-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 6

Author(s):

Chuang-Kai Chueh ◽

Nilanjan Som ◽

Lu-Chu Ke ◽

Meng-Ru Ho ◽

Manjula Reddy ◽

...

Keyword(s):

Active Site ◽

Resting State ◽

Protease Activity ◽

Substrate Binding ◽

Pdz Domain ◽

Structural Basis ◽

Active Site Residues ◽

Specific Protease ◽

Processing Protease ◽

Functional Analyses

ABSTRACT Carboxyl (C)-terminal processing proteases (CTPs) participate in protective and regulatory proteolysis in bacteria. The PDZ domain is central to the activity of CTPs but plays inherently different regulatory roles. For example, the PDZ domain inhibits the activity of the signaling protease CtpB by blocking the active site but is required for the activation of Prc (or Tsp), a tail-specific protease that degrades SsrA-tagged proteins. Here, by structural and functional analyses, we show that in the unliganded resting state of Prc, the PDZ domain is docked inside the bowl-shaped scaffold without contacting the active site, which is kept in a default misaligned conformation. In Prc, a hydrophobic substrate sensor distinct from CtpB engages substrate binding to the PDZ domain and triggers a structural remodeling to align the active-site residues. Therefore, this work reveals the structural basis for understanding the contrasting roles of the PDZ domain in the regulation of CTPs. IMPORTANCE Prc, also known previously as Tsp, is the founding member of the carboxyl-terminal processing protease (CTP) family of PDZ domain-containing proteases that include CtpA and CtpB. The substrate-binding PDZ domain is responsible for regulating the protease activity of CTP proteases; however, the regulatory role of PDZ domain is stimulatory in Prc but inhibitory in CtpA/B. By determining a series of crystal structures of Prc in the unliganded resting state, this study presents the structural basis for PDZ-dependent activation of Prc, the results of which explain the contrasting roles of the PDZ domain in the regulation of the protease activity of CTPs.

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