pykA (PP_1362) encodes one of the two pyruvate kinase isozymes of Pseudomonas putida KT2440 (the other being pykF/PP_4301). Pyruvate kinase (EC 2.7.1.40) catalyzes the final, essentially irreversible step of lower glycolysis: the transfer of the high-energy phosphate from phosphoenolpyruvate (PEP) to ADP, yielding pyruvate and ATP (substrate-level phosphorylation). The enzyme is a cytosolic, homotetrameric protein of the pyruvate kinase family, comprising the characteristic (beta/alpha)8 TIM-barrel catalytic domain, a beta-barrel domain, and a C-terminal regulatory domain. Catalysis requires a divalent cation (Mg2+) and is typically stimulated by a monovalent cation (K+). In pseudomonads, which catabolize sugars predominantly via the Entner-Doudoroff/EDEMP routes rather than a classical complete Embden-Meyerhof-Parnas pathway, pyruvate kinase sits at a key PEP branchpoint, partitioning carbon between pyruvate (feeding acetyl-CoA formation and the TCA cycle) and PEP-consuming biosynthetic routes. Pseudomonas pyruvate kinases are allosterically regulated; the PykA-type isozyme characterized in P. aeruginosa is activated by sugar phosphates such as glucose-6-phosphate.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000287 magnesium ion binding | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Pyruvate kinase catalysis requires a divalent metal cation, canonically Mg2+, which is coordinated in the active site to position the nucleotide phosphates for phosphoryl transfer. This is a conserved, defining feature of the family and is appropriate for this enzyme. Reason: Mg2+ dependence is a family-inferred catalytic feature, but it has not been measured directly for the KT2440 isozyme. Metal binding is subordinate to the exact pyruvate kinase activity and should remain non-core. |
| GO:0004743 pyruvate kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: This is the core molecular function: catalysis of PEP + ADP -> pyruvate + ATP (EC 2.7.1.40). The assignment is robustly supported by family/domain membership (TIGR01064 pyruv_kin, Pfam PF00224/PF02887, the PROSITE pyruvate kinase signature PS00110, and RHEA:18157). Reason: The protein is a full-length member of the pyruvate kinase family with all diagnostic domains and the catalytic-site signature; the reaction is annotated in UniProt (RHEA:18157, EC 2.7.1.40). This is the central function of the gene product. |
| GO:0006096 glycolytic process | IEA GO_REF:0000120 | ACCEPT | Summary: Pyruvate kinase catalyzes the terminal step of the glycolytic (PEP -> pyruvate) sequence, the fifth and final step in the UniPathway "pyruvate from D-glyceraldehyde 3-phosphate" segment (UPA00109). This is the appropriate biological-process annotation for the enzyme. Reason: The PEP-to-pyruvate reaction is by definition part of the glycolytic process; this is the correct and core biological-process term. Note that in P. putida the upper pathway proceeds largely via Entner-Doudoroff/EDEMP, but the pyruvate kinase step itself is still a glycolytic-process reaction. |
| GO:0030955 potassium ion binding | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Many pyruvate kinases are activated by a monovalent cation (typically K+), which binds near the active site and assists catalysis. UniProt lists K+ as a cofactor for this entry, and this annotation is the standard UniRule assignment for the family. Reason: K+ binding is supported by the UniRule assignment and UniProt cofactor record, but it has not been measured directly for the KT2440 isozyme. Retain this family-inferred feature as non-core pending direct evidence. |
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Download this section (compressed HTML)Q: What are the kinetic parameters and allosteric effectors of P. putida KT2440 PykA (PP_1362) specifically, and how do they differ from the second isozyme PykF (PP_4301)?
Experiment: Purify recombinant PP_1362 and determine its substrate kinetics (PEP, ADP), divalent/monovalent cation dependence (Mg2+, K+), oligomeric state, and allosteric activation by sugar phosphates (e.g., glucose-6-phosphate), to confirm the family-inferred cofactor and regulatory annotations in KT2440.
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