Abstract
D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) from Escherichia coli contains two Gly-Gly sequences that have been shown previously to have the characteristics of hinge regions. One of these, Gly(336)-Gly(337), is found in the loop between the substrate binding domain and the regulatory domain. Changing these glycine residues to valine affected the sensitivity of the enzyme to inhibition by L-serine but not the extent of inhibition. The decrease in sensitivity was caused primarily by a decrease in the affinity of the enzyme for L-serine. These mutations also affected the domain rotation of the subunits in response to L-serine binding. A major conclusion of this study was that it defines a minimal limit on the necessary conformational changes leading to inhibition of enzyme activity. That is, some of the conformational differences seen in the native enzyme upon L-serine binding are not critical for inhibition, whereas others are maintained and may play important roles in inhibition and cooperativity. The structure of G336V demonstrates that the minimal effect of L-serine binding leading to inhibition of enzyme activity requires a domain rotation of approximately only 6 degrees in just two of the four subunits of the enzyme that are oriented diagonally across from each other in the tetramer. Moreover the structures show that both pairs of Asn190 to Asn190 hydrogen bonds across the subunit interfaces are necessary for activity. These observations are consistent with the half-the-sites activity, flip-flop mechanism proposed for this and other similar enzymes and suggest that the Asn190 hydrogen bonds may function in the conformational transition between alternate half-the-site active forms of the enzyme.
Highlights
Erative inhibition by L-serine, the end product of its metabolic pathway [1,2,3,4,5]
Previous studies indicated that the double glycine residues at positions 336 and 337, in the loop between the regulatory domain and the substrate binding domain, play a major role in the ability of L-serine to inhibit the activity of PGDH
This was demonstrated by the observation that replacing these residues with those of greater bulk reduced the sensitivity of the enzyme to L-serine. This was interpreted to be the result of a reduction in the rotational flexibility of the main chain bonds in this region. This was further supported by the demonstration that introducing new areas of flexibility adjacent to these mutations could restore and even increase the sensitivity to serine over that of the native enzyme
Summary
Erative inhibition by L-serine, the end product of its metabolic pathway [1,2,3,4,5]. Because the crystal structure of PGDH [6, 7] shows that the loop containing Gly336 and Gly337 does not directly contact other areas of the protein, the introduction of a bulky side chain in this region suggested that hindering the ability of these residues to rotate freely has a significant effect on the ability of the effector to modulate the inhibition of the active site This conclusion is further supported by the observation that introducing additional potential for free rotation about the polypeptide in the form of an additional Gly residue at position 335 increases the ability of serine to inhibit activity. The crystal structures of these mutant enzymes demonstrate that previously observed domain rotations are not required for inhibition of activity by serine to occur
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
