journal article Dec 23, 2025

Phosphorylation of a conserved aspartate in the catalytic site of eukaryotic elongation factor 2 kinase

Protein Science Vol. 35 No. 1 · Wiley
View at Publisher Save 10.1002/pro.70442
Abstract
Abstract

Eukaryotic elongation factor 2 kinase (eEF‐2K) is a member of the α‐kinase family of atypical serine/threonine kinases. eEF‐2K, the only calmodulin‐activated α‐kinase, phosphorylates the ribosome‐associated GTPase, eukaryotic elongation factor 2 (eEF‐2), suppressing translational elongation. α‐kinases, including eEF‐2K, possess catalytic site geometries that are distinct from those of typical kinases, suggesting possible divergence in their phospho‐transfer mechanisms. Unlike typical protein kinases, where chemistry is known to proceed through a sequential mechanism involving a ternary kinase‐substrate‐ATP•Mg
2+
complex, the nature of the chemical step catalyzed by α‐kinases remains poorly defined. Here, multiple orthogonal lines of evidence, including a crystal structure and solution‐state mass spectrometry data, suggest phosphorylation of a catalytically essential aspartate residue (D284) at the eEF‐2K active site. Previous crystallographic evidence of the presence of a phospho‐aspartate at an equivalent position (D766) in the related
Dictyostelium
α‐kinase MHCK‐A strongly suggests that this species represents a conserved active‐site feature in α‐kinase family members, despite their disparate modes of activation. This observation, together with existing kinetics data on eEF‐2K, raises the possibility that phospho‐transfer chemistry in α‐kinases occurs via an ordered stepwise mechanism involving a phospho‐enzyme intermediate, contrasting with typical protein kinases.
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