Role of α-Helix Conformation Cooperating with NH···S Hydrogen Bond in the Active Site of Cytochrome P-450 and Chloroperoxidase:  Synthesis and Properties of [MIII(OEP)(Cys-Helical Peptide)] (M = Fe and Ga)

1998 ◽  
Vol 120 (47) ◽  
pp. 12264-12273 ◽  
Author(s):  
Takafumi Ueno ◽  
Yukihide Kousumi ◽  
Kumiko Yoshizawa-Kumagaye ◽  
Kiichiro Nakajima ◽  
Norikazu Ueyama ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Active Site ◽  
Helical Peptide ◽  
Helix Conformation ◽  
Α Helix ◽  
Cytochrome P 450 ◽  
Peptide M

scholarly journals Conversion of 19-oxo[2β-2H]androgens into oestrogens by human placental aromatase. An unexpected stereochemical outcome

1990 ◽  
Vol 268 (3) ◽  
pp. 553-561 ◽  
Author(s):  
P A Cole ◽  
C H Robinson
Keyword(s):  
Active Site ◽  
Extended Period ◽  
Direct Role ◽  
Methyl Group ◽  
Enzyme Active Site ◽  
Model Studies ◽  
The Third ◽  
Intensive Investigation ◽  
Cytochrome P 450

Aromatase is a cytochrome P-450 enzyme that catalyzes the conversion of androgens into oestrogens via sequential oxidations at the 19-methyl group. Despite intensive investigation, the mechanism of the third step, conversion of the 19-aldehydes into oestrogens, has remained unsolved. We have previously found that a pre-enolized 19-al derivative undergoes smooth aromatization in non-enzymic model studies, but the role of enolization by the enzyme in transformations of 19-oxoandrogens has not been previously investigated. The compounds 19-oxo[2 beta-2H]testosterone and 19-oxo[2 beta-2H]androstenedione have now been synthesized. Exposure of either of these compounds to microsomal aromatase, in the absence of NADPH, for an extended period led to no significant 2H loss or epimerization at C-2, leaving open the importance of an active-site base. However, in the presence of NADPH there was an unexpected substrate-dependent difference in the stereoselectivity of H loss at C-2 in the enzyme-induced aromatization of 19-oxo[2 beta-2H]-testosterone versus 19-oxo[2 beta-2H]androstenedione. The aromatization results for 17 beta-ol derivative 19-oxo[2 beta-2H]-testosterone correspond to about 1.2:1 2 beta-H/2 alpha-H loss from unlabelled 19-oxotestosterone. In contrast, aromatization results for 19-oxo[2 beta-2H]androstenedione correspond to at least 11:1 2 beta-H/2 alpha-H loss from unlabelled 19-oxoandrostenedione. This substrate-dependent stereoselectivity implies a direct role for an enzyme active-site base in 2-H removal. Furthermore, these results argue against the proposal that 2 beta-hydroxylation is the obligatory third step in aromatase action.

scholarly journals High-resolution neutron structure analyses of porcine pancreatic elastase

2014 ◽  
Vol 70 (a1) ◽  
pp. C1216-C1216
Author(s):  
Taro Tamada ◽  
Takayoshi Kinoshita ◽  
Mitsugu Yamada ◽  
Kazuo Kurihara ◽  
Toshiji Tada ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Serine Protease ◽  
Active Site ◽  
Structural Information ◽  
Hydroxyl Group ◽  
Oxyanion Hole ◽  
Pancreatic Elastase ◽  
Neutron Structure ◽  
Porcine Pancreatic Elastase

Elastase is a serine protease classified in the chymotrypsin family, and is attractive target for studies of structure based drug design (SBDD). The structural information including hydrogen positions and hydration will help us to further elucidate the catalytic mechanism of serine protease. To obtain such structural information, we performed the neutron structure analyses of porcine pancreatic elastase (PPE) with and without its inhibitor using diffraction data obtained at a BIX-3 diffractometer in the research reactor JRR-3. The PPE structure in complex with a peptidic inhibitor, which was used to mimic the tetrahedral intermediate state, was determined to 1.65 Å resolution [1]. His57, Asp102, and Ser195 (chymotrypsin numbering) compose the "catalytic triad" conserved in the active site of serine protease. The complex structure determined by neutron crystallography shows that the hydrogen bond between His57 and Asp102 is essentially short but conventional hydrogen bond, not a low-barrier hydrogen bond. In addition, this neutron structure clearly shows that the oxygen of oxopropyl group of the inhibitor is present as an oxygen anion rather than a hydroxyl group, supporting the role of the oxyanion hole in stabilizing the intermediate in catalysis. The neutron structure of PPE without inhibitor determined to 1.9 Å resolution shows that a water molecule and hydroxyl group of Ser195 block to two backbone amides of Gly193 and Ser195, which form oxyanion hole, respectively. This structural information allows us to understand the role of resting state upon the catalytic reaction. Furthermore, the structural change of the active site residues including hydration structure obtained from the comparison between structures with and without inhibitor may help designing potent inhibitors by SBDD.

scholarly journals Role of E270 in pH- and metal-sensitivities of firefly luciferases

2020 ◽  
Vol 19 (11) ◽  
pp. 1548-1558
Author(s):  
V. R. Viviani ◽  
G. F. Pelentir ◽  
G. Oliveira ◽  
A. Tomazini ◽  
V. R. Bevilaqua
Keyword(s):  
Active Site ◽  
Ph Sensitive ◽  
Ph Sensitivity ◽  
Decreasing Ph ◽  
Α Helix ◽  
Conserved Residue ◽  
Active Site Conformation

The substitutions of the conserved residue E270 at the N-terminal of α-helix 10 in pH-sensitive firefly luciferases stabilize a green emitting active site conformation, decreasing pH-sensitivity.

scholarly journals Probing the flavin transfer mechanism in alkanesulfonate monooxygenase system

2018 ◽  
Author(s):  
PV Dayal ◽  
HR Ellis
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Lag Phase ◽  
Monooxygenase System ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Loop Region ◽  
Alkanesulfonate Monooxygenase ◽  
Α Helix

AbstractBacteria acquire sulfur through the sulfur assimilation pathway, but under sulfur limiting conditions bacteria must acquire sulfur from alternative sources. The alkanesulfonate monooxygenase enzymes are expressed under sulfur-limiting conditions, and catalyze the desulfonation of wide-range of alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase. The mechanism for the transfer of reduced flavin in flavin dependent two-component systems occurs either by free-diffusion or channeling. Previous studies have shown the presence of protein-protein interactions between SsuE and SsuD, but the identification of putative interaction sights have not been investigated. Current studies utilized HDX-MS to identify protective sites on SsuE and SsuD. A conserved α-helix on SsuD showed a decrease in percent deuteration when SsuE was included in the reaction. This suggests the role of α-helix in promoting protein-protein interactions. Specific SsuD variants were generated in order to investigate the role of these residues in protein-protein interactions and catalysis. Variant containing substitutions at the charged residues showed a six-fold decrease in the activity, while a deletion variant of SsuD lacking the α-helix showed no activity when compared to wild-type SsuD. In addition, there was no protein-protein interactions identified between SsuE and his-tagged SsuD variants in pull-down assays, which correlated with an increase in the Kd value. The α-helix is located right next to a dynamic loop region, positioned at the entrance of the active site. The putative interaction site and dynamic loop region located so close to the active site of SsuD suggests the importance of this region in the SsuD catalysis. Stopped-flow studies were performed to analyze the lag-phase which signifies the stabilization and transfer of reduced flavin from SsuE to SsuD. The SsuD variants showed a decrease in lag-phase, which could be because of a downturn in flavin transfer. A competitive assay was devised to evaluate the mechanism of flavin transfer in the alkanesulfonate monooxygenase system. A variant of SsuE was generated which interacted with SsuD, but was not able to reduce FMN. Assays that included varying concentrations of Y118A SsuE and wild-type SsuE in the coupled assays showed a decrease in the desulfonation activity of SsuD. The decrease in activity could be by virtue of Y118A SsuE competing with the wild-type SsuE for the putative docking site on SsuD. These studies define the importance of protein-protein interactions for the efficient transfer of reduced flavin from SsuE to SsuD leading to the desulfonation of alkanesulfonates.

scholarly journals Electronic and Steric Control of n→π* Interactions: Stabilization of the α‐Helix Conformation without a Hydrogen Bond

ChemBioChem ◽  
2019 ◽  
Vol 20 (7) ◽  
pp. 963-967 ◽  
Author(s):  
Nicole A. Wenzell ◽  
Himal K. Ganguly ◽  
Anil K. Pandey ◽  
Megh R. Bhatt ◽  
Glenn P. A. Yap ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Π Interactions ◽  
Helix Conformation ◽  
Α Helix ◽  
Steric Control

Heparan sulphate N-sulphotransferase activity: reaction mechanism and substrate recognition

2003 ◽  
Vol 31 (2) ◽  
pp. 331-334 ◽  
Author(s):  
Y. Kakuta ◽  
L. Li ◽  
L.C. Pedersen ◽  
L.G. Pedersen ◽  
M. Negishi
Keyword(s):  
Active Site ◽  
Transfer Reaction ◽  
Substrate Recognition ◽  
Heparan Sulphate ◽  
Critical Role ◽  
Random Coil ◽  
Group Transfer ◽  
Α Helix ◽  
Model Chain

Human heparan sulphate N-deacetylase/N-sulphotransferase 1 sulphates the NH3+ group of the glucosamine moiety of the heparan chain in heparan sulphate/heparin biosynthesis. An open cleft that runs perpendicular to the sulphate donor 3´-phosphoadenosine 5´-phosphosulphate may constitute the acceptor substrate-binding site of the sulphotransferase domain (hNST1) [Kakuta, Sueyoshi, Negishi and Pedersen (1999) J. Biol. Chem. 274, 10673–10676]. When a hexasaccharide model chain is docked into the active site, only a trisaccharide (-IdoA-GlcN-IdoA-) portion interacts directly with the cleft residues: Trp-713, His-716 and His-720 from α helix 6, and Phe-640, Glu-641, Glu-642, Gln-644 and Asn-647 from random coil (residues 640–647). Mutation of these residues either abolishes or greatly reduces hNST1 activity. Glu-642 may play the critical role of catalytic base in the sulphuryl group transfer reaction, as indicated by its hydrogen-bonding distance to the NH3+ group of the glucosamine moiety in the model and by mutational data.

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