The 2.4 Å structure of Zymomonas mobilis pyruvate kinase: Implications for stability and regulation

Meneely, Kathleen M.; McFarlane, Jeffrey S.; Wright, Collette L.; Vela, Kathryn; Swint-Kruse, Liskin; Fenton, Aron W.; Lamb, Audrey L.

doi:10.1016/j.abb.2023.109679

Cited by 2 publications

(18 citation statements)

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“…Thus, stability might be a key factor explaining the differences between hLPYK and ZmPYK. Indeed, WT ZmPYK, with an apparent T m of 50.1 C, is at least 10 C less thermostable than hLPYK (61.4 C) (Meneely et al, 2023). As such, ZmPYK may have a reduced ability to "buffer" against destabilizing substitutions.…”

Section: Possible Origins Of the Wt/inactive Split Phenotypementioning

confidence: 99%

“…The physicochemical scales used in these analyses were: charge and/or polarity, side chain branch location ("branching"), SESA (Bendell et al, 2014), octanol scale ("hydrophobicity") (Wimley et al, 1996), and position-specific helical propensity (Kumar & Bansal, 1998). The helix positions for H48 and F446 (V169 is not in a helix) were determined from the crystal structure (Meneely et al, 2023) using the definitions from Kumar and Bansal (1998). H48 occupies the second position (N2) within its helix and F446 caps the C-terminus of its helix (C-cap).…”

Section: Physicochemical Correlationsmentioning

confidence: 99%

“…(ii) Sequence: ZmPYK and hLPYK share only 37% sequence identity (Page et al, 2022; Steiner et al, 1998); furthermore, ZmPYK lacks the 45 residue N‐terminal extension of hLPYK as well as 9 residues in the C domain that contribute to hLPYK tetramerization (Figure S1) (Swint‐Kruse et al, 2023). (iii) Oligomeric state: Due to the shortened C domain, ZmPYK is dimeric whereas most other PYK isozymes are tetrameric (McFarlane et al, 2019; Meneely et al, 2023; Pawluk et al, 1986). (iv) Allostery: ZmPYK appears to lack the allosteric regulation that is a key functional feature of most other PYK homologs (Munoz & Ponce, 2003; Page et al, 2022; Pawluk et al, 1986; Steiner et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

“…The two homologs also have highly similar K app‐PEP values: ZmPYK's value is 0.13 ± 0.9 mM (Page et al, 2022) and hLPYK's is 0.17 ± 0.02 mM (Swint‐Kruse et al, 2021, 2023). Furthermore, both isozymes have the same monomeric structural architecture; the common regions of their unliganded monomeric structures—comprising three domains that span 475 amino acids—show a root mean square deviation (RMSD) between 0.9 and 1.0 Å (7UEH (Meneely et al, 2023) vs. 6NN7 (McFarlane et al, 2019); Figure S1).…”

Section: Introductionmentioning

confidence: 99%

“…These substitution variants could either lack catalytic activity or lack the stability required for detectable activity. Since all positions with the WT/inactive split outcome were at least 13 Å from the active site (Meneely et al, 2023; Page et al, 2022), we hypothesized that inactive substitution variants did not directly affect catalysis but instead were destabilized. We further hypothesized that, if substitutions at a position modulated both protein function and stability, then decreased protein stability could mask modulatory effects on protein function.…”

Section: Introductionmentioning

confidence: 99%

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Rheostatic contributions to protein stability can obscure a position's functional role

O'Neil,

Swint‐Kruse,

Fenton

2024

Protein Science

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Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing personalized medicine and bioengineering. Current methods cannot accurately predict which proteins contain rheostat positions or their substitution outcomes. To compare the prevalence of rheostat positions in homologs, we previously investigated their occurrence in two pyruvate kinase (PYK) isozymes. Human liver PYK contained numerous rheostat positions that tuned the apparent affinity for the substrate phosphoenolpyruvate (Kapp‐PEP) across a wide range. In contrast, no functional rheostat positions were identified in Zymomonas mobilis PYK (ZmPYK). Further, the set of ZmPYK substitutions included an unusually large number that lacked measurable activity. We hypothesized that the inactive substitution variants had reduced protein stability, precluding detection of Kapp‐PEP tuning. Using modified buffers, robust enzymatic activity was obtained for 19 previously‐inactive ZmPYK substitution variants at three positions. Surprisingly, both previously‐inactive and previously‐active substitution variants all had Kapp‐PEP values close to wild‐type. Thus, none of the three positions were functional rheostat positions, and, unlike human liver PYK, ZmPYK's Kapp‐PEP remained poorly tunable by single substitutions. To directly assess effects on stability, we performed thermal denaturation experiments for all ZmPYK substitution variants. Many diminished stability, two enhanced stability, and the three positions showed different thermal sensitivity to substitution, with one position acting as a “stability rheostat.” The differences between the two PYK homologs raises interesting questions about the underlying mechanism(s) that permit functional tuning by single substitutions in some proteins but not in others.

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Section: Possible Origins Of the Wt/inactive Split Phenotypementioning

confidence: 99%