Substrate access channel

Krainev AG, Weiner LM, Kondrashin SK, Kanaeva IP, Bach-manova GI. 1991. Substrate access channel geometry of soluble and membrane-bound cytochromes P450 as studied by interactions with type II substrate analogues. Arch Biochem Biophys 288 17-21. 

Fig. 5. Stereo view showing the substrate access channel in eNOS. The heme is lightly shaded and the substrate, L-Arg, is darkly shaded. The channel is deep yet solvent accessible for ready entry of substrate and exit of product. Part of the access channel is shaped by the second molecule (shaded) in the dimer. There appears to be no requirement for major structural changes upon substrate binding or release.

Winn PJ, Ludemann SK, Gauges R, et al. Comparison of the dynamics of substrate access channels in three cytochrome P450s reveals different opening mechanisms and a novel functional role for a buried arginine. Proc Natl Acad Sci U S A 2002 99 5361-5366. 

Fig. 5.12 The HRP substrate access channel. The aromatic residues flanking the access channel are highlighted. The arrow, located in the access channel, points to the 5-mew-carbon atom...

Fig. 22. A possible peroxide ghost in the galactose oxidase active site. Two crystallographically well-dehned solvent molecules (HOH 294 and HOH 703, PDB IGOG numbering) lie along the face of the active site metal complex at the base of the substrate access channel in the resting enzyme.

Figure 13 The active-site structure of P450bm-3 with palmitoleic acid bound to the substrate pocket. The fatty acid substrate extends along the substrate access channel to the surface of the protein. The side chains lining the channel are also shown...

E. coli have also been determined (3, 9, 24-26). Figure 1 depicts the polypeptide backbone of the yeast enzyme, indicating the position of the heme, and the proximal and distal histidine residues. The structure can be divided into N- and C-terminal domains, and the heme is in a cavity at the domain interface. The substrate access channel is also at the domain interface and is discussed in Section V. The secondary structure is dominated by a-helices with only a small amount of jS-sheet in the proximal domain. The refined structures of the recombinant wild-type enzymes are essentially identical to that of the yeast enzyme, but small differences are observed in the mutants around the mutated residues (3). 

Fig. 18. Substrate access channel leading to the distal heme cavity in CCP, LIP, ARP, and HRP. The HRP diagram was generated by replacement of the ARP residues with the corresponding residues in HRP (see text). The residues surrounding the channels are represented by their van der Waals surfaces. Adapted from Poulos and Fenna (14) and Kunishima et al. (30).

The structure of the chimeric CYP2C5 retains the overall P450 fold, with the structural elements on the proximal side of the heme well-conserved when compared to the prokaryotic structures. The major differences lie in the elements forming the active site, including those that form the putative substrate access channel. The biggest differences lie in the position of the G helix, and the region from the B helix to the C helix. The G helix adopts a position more elevated from the heme when compared with the bacterial P450cam structure, while the B-G loop has moved away from the N-terminal domain and towards the I helix. The majority of... 

The region 214—227 has been postulated to interact with the membrane and to serve as a substrate-access channel . Mutations in this region yielded some changes in kinetic parameters toward cholesterol. 

Other work has been on membrane topology, and antibody studies indicate that P450 8A1 is mainly exposed on the cytoplasmic site of the endoplasmic reticulum with a single transmembrane anchor . The (imstable) substrate, prostaglandin H2, is produced in the lumen and apparently passes through the membrane to reach P450 8A1. Antibodies raised to the peptides of the putative substrate channel (66-75 and 95-116) interact only after membrane solubilization, implying that the substrate-access channel is very near the membrane . 

K. H. Ruan (2002). Substrate access channel topology in membrane-bound prostacyclin synthase. Biochem. J. 362, 545-551. 

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