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In addition, all UCHs appear to contain a flexible active site cross-over polypeptide loop that regulates entry of the substrate into the active site and to impart its specificity (Popp et al., 2009; Zhou et al., 2012).
This could be due to loss of an interaction with a lumenal region of PS1, thus altering active site conformation, or the fact that these residues normally block the entry of the substrate into the active site.
In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 χ1 rotamer opens the active site to facilitate entry of the substrate.
Up to a point, increasing the positive charge would increase the level of electrostatic attraction and thus accelerate entry of the substrate protein into the pore and strengthen the interaction with the Phe clamp region.
The combination of hinge opening and rotation of Phe31 into the χ1 = 180° rotamer in the hE NADPH holoenzyme opens the active site to facilitate entry of the substrate.
According to Moustafa et al. [ 50], during the binding of the substrate to the catalytic site of the enzyme, His223 assumes conformation A, blocking the entry of oxygen, thus allowing for the entry of the substrate.
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The mobility in this region of the protein could mean that it serves as a gateway in HbpA for entry of the aromatic substrate.
The compounds are expected to bind in a channel that may be used for entry of the nucleotide substrates and exit of the PPi product (Figure 8D) [32].
For this analysis, cells dissociated from the E18 HP and GE were exposed to a hypo-osmotic solution before sorting, to allow the entry of the fluorescent LacZ substrate.
The latter was shown to participate in the chlorite degradation mechanism and to adopt two distinct conformations, one pointing toward the heme iron (here referred to as "in") and one pointing toward the entry of the active site substrate access channel (here referred to as "out").
Furthermore, we could discern no effect of inhibition of the proteolytic activities of the proteasome, nor could we find a role for the Rpt2p proteasome subunit [ 12], which is required for entry of a substrate into the catalytic core of the proteasome barrel [ 47].
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