scholarly journals Effect of replacement of ferriprotoporphyrin IX in the haem domain of cytochrome P-450 BM-3 on substrate binding and catalytic activity

1995 ◽  
Vol 310 (3) ◽  
pp. 939-943 ◽  
Author(s):  
S Modi ◽  
W U Primrose ◽  
L Y Lian ◽  
G C K Roberts
Keyword(s):  
Fatty Acids ◽  
Catalytic Properties ◽  
Substrate Binding ◽  
Dimethyl Ester ◽  
Binding Pocket ◽  
Dodecanoic Acid ◽  
Catalytic Rate ◽  
Cytochrome P 450 ◽  
Haem Iron ◽  
Reductase Domain

Bacillus megaterium cytochrome P-450 BM-3 (coded by gene CYP102) is a catalytically self-sufficient mono-oxygenase, with both cytochrome P-450 and NADPH:cytochrome P-450 reductase domains, that catalyses the hydroxylation of fatty acids. The natural ferriprotoporphyrin IX has been removed from the haem domain of cytochrome P-450 BM-3 by treatment with acidified acetone, and it has been shown that, under carefully controlled conditions, haem can be added back to the resultant apoprotein to obtain a fully reconstituted haem domain with spectroscopic, substrate-binding and catalytic properties indistinguishable from those of the native domain. Replacement of the natural haem with ferriprotoporphyrin IX dimethyl ester yields a protein which has a higher affinity for the substrate dodecanoic acid and (in the presence of the reductase domain) the same catalytic rate as the native haem domain. Replacement with ferrimesoporphyrin IX yields a protein with the same affinity for substrate, but a reduced catalytic turnover. These results suggest that the haem moiety has a role in the creation of the binding pocket for substrate, and that modification of the electron density on the haem iron effects the catalytic rate.

scholarly journals ω-Oxidation impairs oxidizability of polyenoic fatty acids by 15-lipoxygenases: consequences for substrate orientation at the active site

1998 ◽  
Vol 336 (2) ◽  
pp. 345-352 ◽  
Author(s):  
Igor IVANOV ◽  
Kristin SCHWARZ ◽  
Herman G. HOLZHÜTTER ◽  
Galina MYAGKOVA ◽  
Hartmut KÜHN
Keyword(s):  
Fatty Acids ◽  
Active Site ◽  
Substrate Binding ◽  
Salt Bridge ◽  
Binding Pocket ◽  
Substrate Affinity ◽  
Carboxylate Group ◽  
Fatty Acid Binding ◽  
Substrate Orientation ◽  
Binding Cleft

During oxygenation by 15-lipoxygenases, polyenoic fatty acids are bound at the active site in such a way that the ω-terminus of the fatty acids penetrates into the substrate binding pocket. In contrast, for arachidonic acid 5-lipoxygenation, an inverse head to tail orientation has been suggested. However, an inverse orientation may be hindered by the large energy barrier associated with burying the charged carboxylate group in the hydrophobic environment of the substrate binding cleft. We studied the oxygenation kinetics of ω-modified fatty acids by 15-lipoxygenases and found that ω-hydroxylation strongly impaired substrate affinity (higher Km), but only moderately altered Vmax. In contrast, ω-carboxylation completely prevented the lipoxygenase reaction; however, methylation of the additional carboxylate group restored the activity. Arg403 of the human 15-lipoxygenase has been implicated in fatty acid binding by forming a salt bridge with the carboxylate group, and thus mutation of this amino acid to an uncharged residue was supposed to favour an inverse substrate orientation. The prepared Arg403 → Leu mutant of the rabbit 15-lipoxygenase was found to be a less effective catalyst of linoleic acid oxygenation. However, the oxygenation rate of ω-hydroxyarachidonic acid was similar when the wild-type and mutant enzyme were compared, and the patterns of oxygenation products were identical for both enzyme species. These data suggest that introduction of a polar, or even charged residue, at the ω-terminus of substrate fatty acids in connection with mutation of Arg403 may not alter substrate alignment at the active site of 15-lipoxygenases.

Significant improvement to the catalytic properties of aspartate aminotransferase: Role of hydrophobic and charged residues in the substrate binding pocket

Biochemistry ◽  
1994 ◽  
Vol 33 (1) ◽  
pp. 90-97 ◽  
Author(s):  
Eleonore Koehler ◽  
Mark Seville ◽  
Joachim Jaeger ◽  
Ian Fotheringham ◽  
Michael Hunter ◽  
...  
Keyword(s):  
Aspartate Aminotransferase ◽  
Catalytic Properties ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Charged Residues

scholarly journals Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yufei Han ◽  
Qian Zhuang ◽  
Bo Sun ◽  
Wenping Lv ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Biochemical Characterization ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Transmembrane Segments ◽  
Therapeutic Molecules ◽  
Binding Cavity ◽  
Immune System Regulation ◽  
Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

scholarly journals Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes

1996 ◽  
Vol 319 (3) ◽  
pp. 675-681 ◽  
Author(s):  
Jean-Paul RENAUD ◽  
Dmitri R. DAVYDOV ◽  
Karel P. M. HEIRWEGH ◽  
Daniel MANSUY ◽  
Gaston HUI BON HOA
Keyword(s):  
Thermodynamic Parameters ◽  
Protein Interactions ◽  
Substrate Binding ◽  
Principal Component ◽  
Yeast Saccharomyces Cerevisiae ◽  
Human Cytochrome ◽  
Different Temperatures ◽  
Haem Protein ◽  
Cytochrome P 450 ◽  
Spin Transitions

An approach to the quantitative spectral analysis of substrate binding and inactivation of cytochrome P-450 in microsomes is described. The method is based on the application of the principal component analysis technique on the Soret-region spectra measured at different temperatures at various concentrations of substrate. This approach allowed us to study the thermodynamic parameters of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast (Saccharomyces cerevisiae) microsomes. These parameters are discussed in comparison with the values reported earlier by Ristau et al. [(1979) Acta Biol. Med. Ger. 38, 177–185] for rabbit liver cytochrome P-450 2B4 in solution with benzphetamine as a substrate. Our analysis shows the substrate-free states of 2B4 and 3A4 to be very similar. However, substrate binding seems to perturb haem-protein interactions in 3A4 in contrast with 2B4, where the effect of substrate binding on the thermodynamic parameters of spin transitions was insignificant. The implication of the results for the mechanism of substrate-induced spin shift is discussed.

Characterization of the Acyl Substrate Binding Pocket of Acetyl-CoA Synthetase†

Biochemistry ◽  
2006 ◽  
Vol 45 (38) ◽  
pp. 11482-11490 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Barrett I. Woods ◽  
Kerry S. Smith
Keyword(s):  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Acetyl Coa

Structure and mechanism of the γ-glutamyl-γ-aminobutyrate hydrolase SpuA from Pseudomonas aeruginosa

2021 ◽  
Vol 77 (10) ◽  
pp. 1305-1316
Author(s):  
Yujing Chen ◽  
Haizhu Jia ◽  
Jianyu Zhang ◽  
Yakun Liang ◽  
Ruihua Liu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Class I ◽  
Polyamine Metabolism ◽  
Binding Assays ◽  
Starting Point ◽  
Family Activity ◽  
Living Organisms

Polyamines are important regulators in all living organisms and are implicated in essential biological processes including cell growth, differentiation and apoptosis. Pseudomonas aeruginosa possesses an spuABCDEFGHI gene cluster that is involved in the metabolism and uptake of two polyamines: spermidine and putrescine. In the proposed γ-glutamylation–putrescine metabolism pathway, SpuA hydrolyzes γ-glutamyl-γ-aminobutyrate (γ-Glu-GABA) to glutamate and γ-aminobutyric acid (GABA). In this study, crystal structures of P. aeruginosa SpuA are reported, confirming it to be a member of the class I glutamine amidotransferase (GAT) family. Activity and substrate-binding assays confirm that SpuA exhibits a preference for γ-Glu-GABA as a substrate. Structures of an inactive H221N mutant were determined with bound glutamate thioester intermediate or glutamate product, thus delineating the active site and substrate-binding pocket and elucidating the catalytic mechanism. The crystal structure of another bacterial member of the class I GAT family from Mycolicibacterium smegmatis (MsGATase) in complex with glutamine was determined for comparison and reveals a binding site for glutamine. Activity assays confirm that MsGATase has activity for glutamine as a substrate but not for γ-Glu-GABA. The work reported here provides a starting point for further investigation of polyamine metabolism in P. aeruginosa.

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