Role of the Invariant Peptide Fragment Forming NH···S Hydrogen Bonds in the Active Site of Cytochrome P-450 and Chloroperoxidase:  Synthesis and Properties of Cys-Containing Peptide Fe(III) and Ga(III) (Octaethylporphinato) Complexes as Models

1999 ◽  
Vol 38 (6) ◽  
pp. 1199-1210 ◽  
Author(s):  
Takafumi Ueno ◽  
Nami Nishikawa ◽  
Shino Moriyama ◽  
Seiji Adachi ◽  
Keonil Lee ◽  
...  
Keyword(s):  
Hydrogen Bonds ◽  
Active Site ◽  
Peptide Fragment ◽  
Cytochrome P 450

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 An analysis of the role of active site protic residues of cytochrome P-450s: mechanistic and mutational studies on 17α-hydroxylase-17,20-lyase (P-45017α also CYP17)

1998 ◽  
Vol 330 (2) ◽  
pp. 967-974 ◽  
Author(s):  
Peter LEE-ROBICHAUD ◽  
E. Monika AKHTAR ◽  
Muhammad AKHTAR
Keyword(s):  
Hydrogen Bonds ◽  
Active Site ◽  
Catalytic Properties ◽  
Active Form ◽  
Proton Donor ◽  
Wild Type ◽  
Cleavage Reaction ◽  
Acyl Carbon ◽  
Hydroxylation Reaction

Certain cytochrome P-450s involved in the transformation of steroids catalyse not only the hydroxylation process associated with the group of enzymes, but also an acyl-carbon cleavage reaction. The hydroxylation occurs using an iron-monooxygen species while the acyl-carbon cleavage has been suggested to be promoted by an iron peroxide. In this paper we have studied the role of active site protic residues, Glu305 and Thr306, in modulating the two activities. For this purpose, the kinetic parameters for the hydroxylation reaction (pregnenolone → 17α-hydroxypregnenolone) and two different versions of acyl-carbon cleavage (17α-hydroxypregnenolone → dehydroepiandrosterone and 3β-hydroxyandrost-5-ene-17β-carbaldehyde → 3β-hydroxyandrost-5,16-diene+androst-5-ene-3β,17α-diol) were determined using the wild-type human CYP17 and its eight different single and double mutants. In addition the propensity of the proteins to undergo a subtle rearrangement converting the 450 nm active-form into an inactive counterpart absorbing at 420 nm, was monitored by measuring the of the P-450 → P-420 conversion. The results are interpreted to draw the following conclusions. The functional groups of Glu305 and Thr306 do not directly participate in the two proton delivery steps required for hydroxylation but may be important participants for the provision of a net work of hydrogen bonds for ‘activating’ water that then acts as a proton donor. The loss of any one of these residues is, therefore, only partially debilitating. That the mutation of Thr306 impairs the hydroxylation reaction more than it does the acyl-carbon cleavage is consistent with the detailed mechanistic scheme considered in this paper. Furthermore attention is drawn to the fact that the mutation of Glu305 and Thr306 subtly perturbed the architecture of the active site, which affects the geometry of this region of the protein and therefore its catalytic properties.

scholarly journals The cytochrome P-450 active site. Regiospecificity of prosthetic heme alkylation by olefins and acetylenes.

1983 ◽  
Vol 258 (7) ◽  
pp. 4202-4207 ◽  
Author(s):  
K L Kunze ◽  
B L Mangold ◽  
C Wheeler ◽  
H S Beilan ◽  
P R Ortiz de Montellano
Keyword(s):  
Active Site ◽  
Cytochrome P 450
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