Structural Co-optation and Loss-of-function Underlie the Evolution of Regulatory Novelty in the Glucokinase Regulatory Protein

evolutionary biochemistry, small-molecule allostery, glucokinase regulatory protein, glucose homeostasis, protein regulation. This PDF file includes: Main Text Figures 1 to 3 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 10, 2026. ; https://doi.org/10.64898/2026.05.08.723886doi: bioRxiv preprint 2

1 The glucokinase regulatory protein (GKRP) derives from an ancestral etherase. Despite existing 2 as a single locus in the metazoans, GKRP evolved multiple novel functions unrelated to etherase 3 activity. In jawed vertebrates, a protein-protein interaction (PPI) emerged that inhibits glucokinase 4 (GCK) activity in the liver. This PPI is critical to maintaining glucose homeostasis. In mammals, 5 GKRP is allosterically regulated by carbohydrates, with 6-phospharylated sugars promoting 6 inhibition of GCK by GKRP, while 1-phosphorylated sugars relieve inhibition. Here, we use a 7 vertical evolutionary approach to identify the genetic, biochemical, and biophysical mechanisms 8 underlying the emergence of small-molecule allostery in GKRP. We pinpointed a single leucine to 9 valine substitution in the N-terminus of GKRP from the ancestor of the euarchontoglires that, 10 when introduced into the non-regulated placental mammal GKRP ancestor, installed sensitivity to 11 sorbitol-6-phosphate (S6P). Interestingly, GKRP’s inhibitory activity in the absence of S6P was 12 reduced but unchanged in its presence. The mutation enabled co-optation of the ancestral 13 etherase active site, which also existed as an ambiguous phosphorylated carbohydrate binding 14 site in unregulated GKRPs. This substitution likely introduced an alternative conformation of the 15 N-terminus causing apo-GKRP to sample a binding incompetent state prior to GCK binding. Our 16

suggest a simple model of the evolution of protein functional novelty where a single 17 mutation can cause a large functional shift via co-optation of pre-existing structural features. 18 Importantly, in contrast to many models of protein evolution, ours does not require the addition of 19 new genetic material to realize a novel function such as small-molecule allosteric regulation. 20 21 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 10, 2026. ; https://doi.org/10.64898/2026.05.08.723886doi: bioRxiv preprint 3 Main Text 22 23

24 25 The evolution of functional novelty in proteins is essential to organismal diversification (1, 2). 26 Metabolic expansion (3, 4), cell signaling (5, 6), and protein regulation (7, 8) all require the 27 emergence of new protein functions. Current models of the evolution of novel protein functions 28 require the introduction of new genetic material via gene duplication (9–11), horizontal gene 29 transfer (12, 13), or de novo gene birth (14, 15). At the other end of that process lies gene death, 30 in which a previously protein-encoding gene loses its selected function and drifts away, becoming 31 a pseudogene (16). 32 In contrast to these models of functional novelty, the glucokinase regulatory protein (GKRP) 33 appears to have existed as a single locus across eukaryotic evolution (17). Despite the lack of 34 new genetic material, GKRP has evolved at least two novel functions in the vertebrate lineage 35 (18). The first trait to emerge was an inhibitory protein-protein interaction with glucokinase (GCK). 36 In the liver, the synthesis of glucose-6-phosphate by GCK triggers glycogen synthesis (19). When 37 systemic glucose levels are low, GKRP binds to GCK inhibiting its activity (20, 21). Additionally, 38 the GCK-GKRP complex is translocated to the nucleus, removing GCK activity from the 39 cytoplasm (22). Recently, Kamalaldinezabadi et al. demonstrated that this regulatory, heteromeric 40 interaction between GKRP and GCK evolved in the ancestor of the gnathostomes (jawed 41 vertebrates) via exaptation of a hydrophobic patch in GCK and the insertion of a binding loop into 42 GKRP that fits into the GCK patch (18). 43 The second new function to emerge was allosteric regulation of GKRP’s inhibitory activity by 44 phosphorylated carbohydrates, a characteristic that is observed in extant mammals. In the 45 unliganded state (23), an N-terminal extension in GKRP adopts a folded conformation distal to 46 the GCK binding site at the junction of its constituent lid and dual sugar-isomerase domains (SIS; 47 Fig. 1A). In the bound state (20), the N-terminus an extended conformation where it can interact 48 with residues at the GCK binding interface. In rats and humans, phosphorylated carbohydrates 49 such as fructose-6-phosphate (F6P) and sorbitol-6-phosphate (S6P) promote the GKRP-GCK 50 interaction and enhance inhibition. Conversely, fructose-1-phosphate (F1P) inhibits the GKRP-51 GCK interaction. Intriguingly, the GKRP from the frog Xenopus laevis is insensitive to allosteric 52 regulation by phosphorylated carbohydrates despite the structure of the frog GKRP-GCK complex 53 (24) being superimposable with the mammalian structure, including the positioning of bound 54 ligands. 55 Eukaryotic GKRP evolved from a duplication and uneven fusion of a bacterial gene, murq (17), 56 that encodes a homodimeric bacterial etherase involved in cell wall recycling (Fig. 1B) (25, 26). 57 The structure of the MurQ homodimer (27) is superimposable with mammalian GKRP, including 58 the active site where a MurQ inhibitor overlaps with S6P in the mammalian structure. The GKRP 59 homolog from the excavate amoeba Naegleria gruberi is closer in sequence to vertebrate GKRPs 60 than to MurQ but displays etherase activity comparable to bacterial homologs (17). GKRP has no 61 other known role in N. gruberi, suggesting that GKRP plays some potential role related to MurQ-62 like activity in the amoeba and related organisms, likely digestion of bacterial cell walls (17). 63 Strangely, frog GKRP also displays etherase activity, albeit diminished by multiple orders of 64 magnitude, despite no known role for MurQ-like activity in vertebrates. 65 We used phylogenetics and genomic analysis to determine orthology and synteny in eukaryotic 66 GKRPs. We measured etherase activity and GCK inhibition in extant GKRPs as well as extinct, 67 ancestral GKRPs that we resurrected in the lab. Combined with structural and mutational 68 analysis, we uncovered the genetic, biochemical, and biophysical mechanisms underlying the 69 evolution of functional novelty in GKRP as it transitioned from an active etherase to a member of 70 a protein-protein inhibitory complex that is allosterically regulated by small molecules. Our results 71 demonstrate a model for the evolution of functional novelty that does not require the addition of 72 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 10, 2026. ; https://doi.org/10.64898/2026.05.08.723886doi: bioRxiv preprint 4 new genetic material but instead relies on co-optation and repurposing of protein features 73 encoded at an already existing locus. 74 75

and Discussion 76 77 GKRP is a single locus among Metazoa 78 The functional transition of GKRP to a regulatory protein occurred during vertebrate evolution, 79 specifically between the last common ancestor of vertebrates and the ancestor of the jawed 80 vertebrates (18). The phylogeny of Kamalaldinezabadi et al. shows GKRP as a single locus 81 across all eukaryote evolution. This suggests that no gene duplication event occurred during the 82 evolution of eukaryotic GKRP, which could have provided the genetic raw material to facilitate the 83 functional novelty found in GKRP in the jawed vertebrates and the small-molecule allostery found 84 in mammals. To determine if this was a potential sampling artifact from the previously estimated 85 GKRP phylogeny (18), we investigated GKRP genomic loci among eukaryotes. 86 Ensembl (28) and Genomicus (29) databases enable analysis of relationships of orthology, 87 paralogy, and synteny through gene structure alignments and gene order alignments, 88 respectively, integrated with gene trees. Among chordates and tunicates, Ensembl shows a 89 single GKRP ortholog with highly conserved exon-intron architecture (Fig. S1). In the jawed 90 vertebrates, Genomicus output indicates these are syntenic orthologs (Fig. S2 & S3). The 91 Genomicus output demonstrates the orthology of GKRPs in vertebrates, tunicates, and 92 cephalochordates (Fig. S2 and S3), as well as linking these orthologs to the invertebrate 93 metazoans (Fig. S4). Among the invertebrates, there are lineage specific events, i.e. duplications 94 in a species of sea urchin and a species of ribbon worm, and a gene split event in an anemone 95 (Fig. S5). However, Metazoan GKRPs are generally single locus orthologs. 96 Among Protista, there was at least one gene duplication event prior to the divergence of Metazoa 97 in the ancestor of Apusomonididae and Holozoa (Fig. S6). We constructed a neighbor-joining 98 GKRP phylogeny with amino acid sequences to estimate the relatedness of metazoan GKRPs to 99 the protist paralogs. Our GKRP tree shows that the Metazoan GKRPs cluster with only one of 100 these early GKRP paralogs (Fig. 1D). Independent gkrp losses in Capsaspora and the ancestor 101 of Metazoa supports our contention that GKRP has existed as a single ortholog since the 102 divergence of animals, long before the novel functions emerged in the jawed vertebrates (Fig. 2A) 103 (18). 104 Non-mammalian GKRPs are active etherases 105 The inhibitory interaction between GKRP and GCK is specific to gnathostomes, the jawed 106 vertebrates (Fig. 2A). Extant cephalochordate and tunicate GKRP orthologs do not inhibit their 107 corresponding GCKs (18). This correlates with the phenotype seen in the chordate and vertebrate 108 ancestral GKRPs, which also do not inhibit their corresponding GCKs. As the ancestral function 109 of GKRPs is etherase activity, we tested if etherase activity was compatible with the evolution of 110 the GKRP-GCK interaction along the vertebrate trajectory. We measured etherase activity in both 111 extant GKRPs, and the extinct ancestral GKRPs from Kamalaldinezabadi et al.using a linked 112 enzyme assay coupling hydrolysis of the N-acetylmuramic acid 6-phosphate (MurNAc-6P) lactyl 113 ether bond to production of D-lactate, which is coupled to formazan production. As a control and 114 to provide a benchmark for potential etherase activity, we measured the activity of the extant 115 MurQ from Escherichia coli and found that it displays strong etherase activity and is subject to 116 substrate inhibition (3140 ± 531 M-1 s-1; Ki = 3.36 ± 0.75 mM; Fig. S7; Table S1). 117 A representative activity assay of the extant GKRP from the cephalochordate lancelet 118 demonstrates that etherase activity is retained in non-vertebrate GKRPs, albeit without substrate 119 inhibition (1493 ± 166 M-1 s-1; Fig. 2B; Fig. S8; Table S1). In contrast, rat GKRP lacks detectable 120 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 10, 2026. ; https://doi.org/10.64898/2026.05.08.723886doi: bioRxiv preprint 5 etherase activity (Fig. 2C; Fig. S9; Table S1). Assays of extinct GKRPs demonstrate that the 121 chordate (703 ± 66 M-1 s-1; Fig. 2D; Fig. S10; Table S1) and vertebrate (86 ± 6 M-1 s-1; Fig. 2D; 122 Fig. S11; Table S1) ancestral GKRPs retained etherase activity. Further, the gnathostome 123 ancestral GKRP also displays relatively strong etherase activity (105 ± 13 M-1 s-1; Fig. 2D; Fig. 124 S12; Table S1) demonstrating that enzymatic activity and formation of the GKRP-GCK interaction 125 are not mutually exclusive. This comports with the previous report of etherase activity in GKRP 126 from the frog Xenopus laevis (17). 127 In addition to displaying etherase activity, Veiga-da-Cunha et al. demonstrated that the etherase 128 activity of frog GKRP is inhibited by F6P and most strongly by S6P (17). In contrast, F1P does not 129 inhibit the etherase activity. Those authors were able to measure small amounts of etherase 130 activity in rat GKRP and found that it is similarly inhibited by F6P and S6P. Interestingly, it is also 131 strongly inhibited by F1P. As stated above, structural data indicates that phosphorylated 132 carbohydrates bind at the etherase active site (20, 24). These observations support a model in 133 which the GKRP etherase active site likely existed as an ambiguous phosphorylated 134 carbohydrate binding site before the allosteric modulation of GKRP inhibitory activity emerged. 135 Mechanistic evolution of small-molecule allosteric regulation in GKRP 136 To determine when along the vertebrate evolutionary trajectory allosteric activation of GKRP 137 inhibitory activity (IC50) by phosphorylated carbohydrates first emerged, we measured inhibition of 138 corresponding GCKs by extant and resurrected ancestral GKRPs in the presence and absence of 139 S6P (Table S2). The GKRP from lancelet was previously shown to not inhibit lancelet GCK in the 140 absence of S6P (18). Addition of S6P does not induce inhibitory activity in the lancelet pair (Fig. 141 S13). Frog GKRP inhibits frog GCK with a sub-micromolar IC50 (Fig. S14) and S6P has no effect 142 on the inhibitory activity (Fig. S15). Our resurrected ancestral GKRPs show a similar pattern. The 143 chordate and vertebrate ancestral GKRPs do not inhibit chordate and vertebrate GCKs (18), 144 respectively, and addition of S6P causes no change (Fig. S16 & S17). The gnathostome and 145 tetrapod ancestral GKRPs inhibit their corresponding GCKs (Fig. S18 & S19). Addition of S6P 146 does not alter their inhibitory activity (Fig. S20 & S21). Our measurements suggest that small-147 molecule allostery in GKRP is a mammal specific trait, which is consistent with previous 148 comparative observations (17, 30). 149 Among mammals, allosteric modulation of GKRP inhibition of GCK by phosphorylated 150 carbohydrates has been demonstrated in rabbit, rat, and human (30). Expanding upon these 151 findings, we measured the GKRP inhibition of GCK in proteins from the wombat Vombatus 152 ursinus, a marsupial. Wombat GKRP inhibited its corresponding GCK with a micromolar IC50 153 value similar to the gnathostome and tetrapod ancestral GKRPs (Fig. S22). Addition of S6P does 154 not alter this IC50 value (Fig. S23). Stepwise resurrection of ancestral mammalian GKRPs 155 revealed the emergence of small-molecule allosteric regulation. The inhibitory activity of the 156 GKRP corresponding to the ancestor of placental mammals (M1; Fig. 3A) is insensitive to S6P 157 (Fig. S24 & S25). Resurrection and characterization of the ancestor of the euarchontoglires (M2; 158 Fig. 3A) — the clade containing rabbits, rodents, and primates — shows that small-molecule 159 allostery is specific to this group, as M2 GKRP displays sensitivity to S6P (Fig. S26 & S27). 160 The N-terminus of GKRP (residues 1-30 in human GKRP numbering) is the structural feature that 161 likely modulates the GCK binding interface in response to phosphorylated carbohydrate binding 162 (31). We compared the frog, M1, M2, and human GKRP sequences to determine if there are 163 substitutions in the N-terminus that are responsible for small-molecule allostery. Only eight 164 residues show variation among the four GKRPs, while the rest are conserved (Fig. 3B). Of these 165 eight, only one residue has a substitution between M1 and M2 — an L to V substitution at position 166 28. We introduced the M2 residue into the non-allosterically regulated M1 background. 167 Measurement of GCK inhibition demonstrates that the resulting loss of a single methylene group 168 via the L28V substitution is sufficient to introduce sensitivity to S6P (Fig. 3C; Fig. S28 & S29). 169 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 10, 2026. ; https://doi.org/10.64898/2026.05.08.723886doi: bioRxiv preprint 6 Counterintuitively, the L28V substitution imparts allostery through the loss of inhibitory function in 170 the apo-state of GKRP while leaving activity in the presence of S6P unchanged (Fig. 3C). In the 171 rat-human GKRP-GCK complex, ligand binding to GKRP kinetically stabilizes the GKRP-GCK 172 bound state by increasing the rate constant for forming the encounter complex (kon) and by 173 decreasing the rate constant for dissociation (koff) (32). Based on structural data, the rat GKRP N-174 terminus samples at least two conformations (31). In the S6P bound, inhibitory-competent state, 175 the N-terminus is extended where it can influence residues at the GCK binding interface, likely by 176 stabilizing them in the binding competent state (Fig. 1A). In the F1P bound, inhibitory-incompetent 177 state, the N-terminus is folded at the interface of the LID and SIS domains, far removed from the 178 GCK binding site. We postulate that the L28V substitution affords access to this second 179 conformation by ordering the N-terminus in the apo-state. Martinez et al. produced a truncated rat 180 GKRP missing the first twenty residues (31). This version of rat GKRP loses inhibitory activity in 181 the presence of S6P while activity in the absence remains unchanged. Eliminating the ability to 182 sample the two different N-terminal conformations removes allosteric modulation, providing 183 biochemical and biophysical mechanisms for the emergence of small-molecule allostery 184 consistent with our postulate. 185 186

187 188 Our vertical approach allowed identification of a functional transition point along the GKRP 189 evolutionary trajectory. A single loss-of-function cytosine to thymine substitution in the GKRP 190 gene could lead to the L28V amino acid substitution, which is sufficient to cause co-optation of a 191 pre-existing binding site for phosphorylated carbohydrates derived from a vestigial etherase 192 active site. This suggests that the simplest genetic mechanism can enable ligand-mediated 193 allosteric regulation of proteins. Importantly, the evolution of this regulatory novelty in GKRP did 194 not require the addition of new genetic material. Due to its simplicity, we speculate that our simple 195 model of the evolution of novel protein function is likely to be a general mechanism. 196 197

198 199 Synteny Analysis 200 We used the Ensembl (28) and Genomicus (29) databases to assess orthology and synteny in 201 eukaryotic GKRPs. The GKRP phylogenetic tree (SI file) was estimated using the Neighbor-202 Joining method from a MUSCLE (33) alignment of GKRP amino acid sequences (SI file), all 203 implemented in MEGA 7 (34). 204 205 Activity Assays 206 Extant and extinct recombinant GKRPs and GCKs were produced as described previously (18). 207 Recombinant E. coli MurK was expressed with a C-terminal hexahistidine tag. Recombinant E. 208 coli MurQ was expressed with an N-terminal hexahistidine tag. Both were purified with Ni-NTA 209 affinity chromatography and size-exclusion chromatography. 210 211 GKRP inhibition of GCK was measured as previously described (18). Extinct and extant GCKs 212 were mixed with corresponding GKRPs at varying GKRP concentrations. The protein mixture was 213 incubated for five minutes, with or without S6P, prior to initiation of the GCK reaction. GCK 214 activity was followed by linked enzyme assay where production of glucose-6-phosphate by GCK 215 is coupled to the reduction of NADP+ by glucose-6-phosphate dehydrogenase and measured in a 216 spectrophotometer at 340 nm. 217 218 Etherase activity of E. coli MurQ and extinct and extant GKRPs was measured with a three-step 219 linked enzyme assay (35). First, the etherase catalyzes the hydrolysis of the MurNAc-6P lactyl 220 ether bond, producing GlcNAc-6P and D-lactate. Next, D-lactate dehydrogenase (D-LDH) 221 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 10, 2026. ; https://doi.org/10.64898/2026.05.08.723886doi: bioRxiv preprint 7 catalyzes the transfer of D-lactate’s hydroxyl hydrogen to NAD+, producing pyruvate and NADH. 222 In the last step, diaphorase catalyzes the reduction of p-iodonitrotetrazolium violet (INT) by 223 NADH, recycling NADH into NAD+ and producing formazan, which absorbs at 500 nm. 224 225 The etherase substrate MurNAc-6P was produced by phosphorylation of commercially available 226 N-acetylmuramate by MurK. MurNAc-6P was purified with HILIC HPLC (Fig. S30) on a Shimadzu 227 HPLC system (Shimadzu, Kyoto, Japan) based on a published protocol (35) and its identity 228 confirmed via mass spectrometry (Fig. S31). Various concentrations of D-lactate (134 μ M–900 229 μ M) were assayed in the absence of GKRP or MurQ. A standard curve for D-lactate was 230 constructed based upon the maximum absorbance reached after 15 minutes of incubation. The 231 data were fit to a linear equation of y = ax + b, where y is the maximum absorbance, x is the D-232 lactate concentration, a is the slope of the line, and b is the line’s y-intercept. This assay was 233 repeated with 4 μ L of a MurNAc-6P sample that had been diluted 1:100 with sodium phosphate 234 buffer (40 mM, pH 12.5) and incubated at 37° C overnight. The maximum absorbance obtained 235 was used in the standard curve’s linear equation (y = 1.952x – 0.007253) to calculate the 236 concentration of D-lactate produced, which was then used to calculate the concentration of 237 MurNAc-6P in the original stock solution. Data points for the standard curve and the MurNAc-6P 238 sample were collected in triplicate. 239 240 Full experimental details can be found in SI Materials and Methods. 241 242 Acknowledgments 243 244 Research reported in this publication was supported by the National Institute of General Medical 245 Sciences of the National Institutes of Health under Award Number R01GM133843 (B.G.M.) The 246 content is solely the responsibility of the authors and does not necessarily represent the official 247 views of the National Institutes of Health. Additional funding was provided by the FSU Council on 248 Research and Creativity (B.G.M). 249 250

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Maye r, Enzy matic synthesis and semi-pre para tive iso lation of 332 N-acetylmuramic acid 6-phospha te . Carb ohy dr. Res. 445 , 98–103 (2017). 333 334 335 336 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 10, 2026. ; https://doi.org/10.64898/2026.05.08.723886doi: bioRxiv preprint 10 Figures 337 338 339 Figure 1. GKRP structure, function, and evolution. (A) GKRP (left; pdb:4BB9) consists of two SIS 340 domains (shades of orange) and a LID domain (yellow). GKRP (right; pdb:4LC9) binds to GCK in 341 a cleft between its large (gray) and small (light blue) domains inhibiting GCK’s ability to 342 phosphorylate glucose. Binding of GCK is associated with a rearrangement of the GKRP N-343 terminus (residues 1-30; green spheres). In mammals, GKRP inhibition of GCK is allosterically 344 modulated by phosphorylated carbohydrates with sorbitol-6-phosphate (S6P) promoting GKRP-345 GCK complex formation. Insets show a zoomed view of N-terminus secondary structure. (B) 346 MurQ (pdb:4LZJ) is a bacterial etherase that hydrolyses MurNAc-6P during cell wall recycling. It 347 functions as a homodimer (dark blue and purple). (C) GKRP evolved from a duplication and 348 unequal fusion of two copies of MurQ. (D) Analysis of GKRP synteny among eukaryotes 349 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 10, 2026. ; https://doi.org/10.64898/2026.05.08.723886doi: bioRxiv preprint 11 demonstrates that after a gene duplication event in the Apusomonad ancestor, one copy was lost 350 prior to the divergence of Metazoa, among which GKRP generally exists as a single locus (see 351 SI). 352 353 354 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 10, 2026. ; https://doi.org/10.64898/2026.05.08.723886doi: bioRxiv preprint 12 355 Figure 2. The emergence of GKRP inhibition of GCK in vertebrates and allosteric regulation of 356 GKRP activity in mammals correlates with a loss of ancestral etherase activity. (A) Eukaryotic 357 GKRP phylogeny displaying functional and regulatory properties of extant and extinct GKRPs 358 (phylogeny adapted from (18); a – data from (18); b – data from (30). (B) Representative etherase 359 activity curve showing Michaelis-Menten kinetics for Lancelet GKRP. Data points represent the 360 average observed rates from three technical replicates. Error bars represent standard deviation. 361 (C) Representative etherase activity measurements for Rat GKRP showing no ability to hydrolyze 362 MurNAc-6P. Data points represent the average observed rates from three technical replicates. 363 Error bars represent standard deviation. (D) Specificity constants for MurNAc-6P for extant and 364 extinct MurQ and GKRPs show the loss of etherase activity across GKRPs evolution. Bars 365 represent the average of two biological replicates. Error bars represent standard deviation. 366 367 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 10, 2026. ; https://doi.org/10.64898/2026.05.08.723886doi: bioRxiv preprint 13 368 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 10, 2026. ; https://doi.org/10.64898/2026.05.08.723886doi: bioRxiv preprint 14 Figure 3. Allosteric regulation of GKRP inhibition of GCK evolved via a loss-of-function 369 substitution in the Euarchontoglires ancestor. (A) Measurements of extant and extinct GKRPs’ 370 inhibition of GCK in the absence (-) and presence (+) of sorbitol-6-phosphate (2 mM) along the 371 vertebrate evolutionary trajectory. A functional shift in sensitivity to phosphorylated carbohydrates 372 occurred between the ancestor of placental mammals (M1) and the ancestor of the 373 euarchontoglires (M2). Bars represent the average of two biological replicates. * - indicates 374 significant difference, p < 0.05. a – data from this study; b – from (30). (B) The N-terminus of 375 GKRP (residues 1-30) is highly conserved among tetrapods. Positions with amino acid 376 substitutions are shown. There is a single substitution between the M1 and M2 ancestors. (C) 377 The L28V substitution in the M1 GKRP ancestor background introduces allosteric regulation by 378 reducing the inhibition activity in the absence of phosphorylated carbohydrate while leaving 379 activity unchanged in the presence. Bars represent the average of two biological replicates. See 380 SI Table 2 for error estimates. * indicates significant difference, p < 0.05. 381 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 10, 2026. ; https://doi.org/10.64898/2026.05.08.723886doi: bioRxiv preprint