Site-directed alterations to the geometry of the aspartate transcarbamoylase zinc domain: Selective alteration to regulation by heterotropic ligands, isoelectric point, and stability in urea

Abstract

Structural aspects requisite for allosteric function in the regulatory chain of aspartate transcarbamoylase were explored by site-specific amino acid insertion or substitution within the zinc domain in the region of contact between the catalytic and regulatory chains. Amino acid substitution at two positions yielded enzymes which retained a maximum velocity similar to that of the wild-type enzyme but responded differently from the native enzyme in the presence of regulatory nucleoside triphosphates. A change of zinc coordinate amino acid C109 to histidine and a change of E119 to aspartic acid resulted in enzymes which demonstrated synergistic inhibition by CTP and UTP but not inhibition by CTP in either phosphate buffer or a morpholino-based tri-partate (TP) buffer at pH 7. At pH 8.3, where there is a higher proportion of T-state conformers in the native enzyme, the mutants diverged from their similar kinetic behavior. C109H remained an enzyme which was not inhibited by CTP but was still inhibited by CTP+UTP. E119D was inhibited by both CTP and CTP+UTP. Activation of the mutants by ATP was found to vary either with pH or with phosphate as a buffer component. C109H was activated by ATP in phosphate, while in TP at either pH 7 or 8.3 its activation by ATP was diminished or absent. E119D was activated by ATP in phosphate at pH 7 or in TP at pH 8.3, but not in TP at pH 7. In TP at pH 7, where neither mutant was activated by ATP, the S values and Hill coefficients of the unliganded mutant enzymes resembled those of the ATP-liganded wild-type enzyme. While neither mutation would be predicted to alter the net charge of the holoenzyme, differences in the isoelectric point of the mutants were observed if phosphate was present. This result suggests that the isoelectric point of aspartate transcarbamoylase is conformationally dependent and that the mutants exist in an altered conformation. In addition, the stabilities of both mutant holoenzymes were reduced substantially from those of the wild-type enzyme in 4 M urea. C109H was more stable at pH 8.25 in a Tris buffer; E119D was more stable at pH 7 in the phosphate buffer. Potential effects of these mutations on the active site chemistry and geometry are discussed.

Simufilam Reverses Aberrant Receptor Interactions of Filamin A in Alzheimer’s Disease

“…A region of FLNA unfolds under forces as low as 10 pN [ 17 ], and stress-induced conformational changes have been hypothesized to play a direct role in signaling, either by disrupting existing interactions or inducing new ones [ 18 ]. In an altered conformation implied by a shift in isoelectric focusing point [ 8 , 10 , 19 ] and a change in solubility [ 16 ], FLNA appears to be a critical and deviant receptor-associated protein underlying multiple facets of AD pathology [ 9 , 10 ]. Specifically, deviant FLNA linkages are critical to Aβ 42 -induced tau hyperphosphorylation, leading to neurodegeneration, and to Aβ 42 -induced activation of TLR4, leading to neuroinflammation [ 9 , 10 ].…”