Substrate exit channel

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.

Other oxidases also derive function from bidirectional PCET pathways at the enzyme active site. The recent crystal structures of PSII [206, 207] support suggestions that as the oxygen evolving complex (OEC) steps through its various S-states [208, 209], substrate derived protons are shuttled to the lumen via a proton exit channel, the headwater of which appears to be the Dgi residue hydrogen-bonded to Mn-bound water [210]. The protons are liberated with the proton-coupled oxidation of the Mn-OH2 site. As shown by the structure reproduced in Fig. 17.23, Dgj is diametrically opposite to Y, which has long been known [148, 151, 152] to be the electron relay between the PS II reaction center and OEC. Notwithstanding,... 

The X-ray structures of several human HDACs have been solved and reveal a catalytic mechanism akin to other metalloenzymes that carry out amide hydrolysis (Figure 4.3). The acetyl-lysine substrate is positioned within a narrow channel that ends with a catalytic zinc cation. The metal serves to activate a water molecule to make it more nucleophilic and simultaneously coordinates to the acetyl group to make it a better electrophile. The attack of water results in a tetrahedral intermediate that collapses to give lysine and acetate, the latter leaving through an exit channel separate from the substrate binding pocket... 

Ludemann SK, Lounnas V, Wade RC. How do substrates enter and products exit the buried active site of cytochrome P450cam 1. Random expulsion molecular dynamics investigation of ligand access channels and mechanisms. J Mol Biol 2000 303 797-811. 

Three channels have been implicated as potentially having a role in the access to the active site of CAT. The perpendicular (relative to the plane of the heme) or main channel has been considered as an access channel. The lateral or minor channel has been shown to be important, possibly as an inlet channel, in HPII and in small-subunit enzymes. A third channel has been identified, leading from the heme to the central cavity of the tetramer [200]. All three channels are quite narrow, particularly as they approach the heme-containing active-site cavity, and this restricts accessibility to relatively small molecules, generally not much larger than H2O2. Subtle differences in the size and shape of the channels may influence substrate entry or product exit, contributing to the differences in the reaction rates between different CATs [201]. 

A potential substrate entryway (which presents anphipathic residues possibly to accommodate polar substrate head groups towards the FAAH active site) has been identified next to a-18 and a-19 helices, and it may indicate direct connection between the FAAH active site and the hydrophobic membrane bilayer. The mode for membrane binding of FAAH may facilitate movement of the FAA substrates directly from the bilayer to the active site, with no need for transport of these lipids through the aqueous cytosol. In this model, the substrate would first enter via the membrane to the active site following hydrolysis, the released fatty acid (hydrophobic) and amine (hydrophilic) products would then exit through the membrane-access and cytosolic-access channels, respectively. Moreover, the cytoplasmic port may serve the additional function of providing a way for a water molecule required for deacylation of the FAA-FAAH acyl-enzyme intermediate, which has been already characterized by LC-MS (Patricelli and Cravatt., 1999). 

Steady-state kinetic analysis afforded evidence for intermediate channeling in the lumazine synthase/ riboflavin synthase complex. Briefly, the conversion of 6,7-dimethyl-8-ribityllumazine molecules that have been newly formed by the lumazine synthase module of the protein complex are more rapidly converted to riboflavin than molecules from the bulk solvent. The topological constraint by the capsid is believed to cause this phenomenon. It has been proposed that the channels along the five-fold axes could serve as port of entry and exit for substrates and products. ... 

Cytochrome c peroxidase (CcP) [215] isolated from yeast mitochondria uses cytochrome c(//) as electron donor. Its crystal structure has been determined [216]. It is a monomer of molecular mass 34000 containing a single h-type heme group that is largely buried within the molecule. The Fe is coordinated on one side by a histidine residue while the sixth position is occupied by a weakly bound HjO in a channel suitable for entry and exit of small substrates. 

Because cavity II is wholly internal, the entry and exit for the fatty acid are difficult to identify. Boyington et al. [158] propose that the substrate enters the cavity after rearrangement of the Met-341 and Leu-480 side chains that block the entry. In addition, rearrangement of the side chains of Arg-707 and Val-354, that make a 3.5 A contact, would be necessary to allow the substrate to reach the iron. Minor et al. [62] agree on the part of cavity II near the iron (Fig. 6, cavity Ila), but suggest that the part of the cavity II between Met-341/Leu-480 and Arg-707 (cavity Ilb) may not be suitable for transport of the substrate. They propose three possible channels (not shown) that reach cavity Ila at the opposite side of that proposed by Boyington et al. [ 158]. 

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