Introduction

Isocitrate dehydrogenase kinase/phosphatase (IDHK/P or AceK) is an E. coli enzyme which phosphorylates or dephosphorylates isocitrate dehydrogenase (IDH) on a specific serine residue () thus regulating its activities. This is a regulatory mechanism which enables bacteria to bypass the Krebs cycle via the glyoxylate shunt in response to nutrient availability. AceK is expressed from a single gene, aceK. Both kinase and phosphatase activities reside on the same polypeptide and share the same active sites^[1][2].

Structure^[3]

The AceK structure contains two functional domains: a Kinase domain (KD) where the kinase, phosphatase and ATPase reactions occur, and a regulatory (RD) that helps form allosteric binding pockets involved in regulating the catalytic domain’s function. The is situated in a pocket at the interface between the KD and RD and acts as a bridge.

The , which comprises the amino-terminal half of the AceK sequence, is mainly composed of a-helices and hairpin structures.This domain represents a unique protein fold with no structural homologues. The regulatory domain is linked to the kinase domain by a 27-residue-long a-helix. The , which makes up the carboxy-terminal half of AceK, has a classic bi-lobe protein kinase fold with the located at the interface between the two lobes. The N-terminal lobe consists mainly of a twisted, five-stranded, antiparallel b-sheet and two a-helices. The larger C-terminal lobe is predominantly a-helical with some stretches of antiparallel b-strands. The ATP molecule is under the cover of the five-stranded b-sheet and is shielded by . Loop-b3aC shifts upwards or downwards to controls access to the ATP-binding site. The (SRL) stretches out of the C-terminal lobe. This loop, together with loop-b3aC, forms a large cleft that is the .

Function

AceK monitors general metabolism by responding to the levels of a wide variety of metabolites. This ability of AceK allows the IDH phosphorylation cycle to compensate for substantial perturbations of the system^[1]. When a less preferred carbon source is available, the cell responds by phosphorylating IDH, thus inactivating IDH and activating the glyoxylate bypass^[2]. Many of the regulatory effectors are derived from the end products of the glyoxylate bypass, and represent negative feedback inhibition mechanisms^[4].

Furthermore, Depletion in AMP levels signals that the cell requires energy and isocitrate will continue through the Krebs cycle with IDH dephosphorylated^[4].AMP binds directly to AceK, activate IDH phosphatase and inhibit both IDH kinase and the intrinsic ATPase activities^[5]. An AMP-mediated conformational change exposes and shields ATP, acting as a switch between AceK kinase and phosphatase activities, and IDH-binding induces further conformational change for AceK activation. During the activation SRL of the kinase domain recognizes the IDH active cleft and inserts into a binding pocket formed by the ICDH dimer, yielding strict substrate specificity and triggering substrate conformational change for catalysis which allow the Ser113 residue be more accessible for AceK^[3].

Active Site

A catalytic triad is present in AceK, involving residues Asp457, Asn462 and Asp475, in the ATP binding region^[4]. Asp477 and Asp475 interact with the g-phosphate of ATP, and Asp475 coordinates the ATP-liganded single magnesium ion. This signature motif is crucial for the kinase activity^[3]. Lys461 interacts with ATP and Ser113, playing a significant role in binding of the reactants and keeping them in close contact conformation. Lys336, a key residue for ATP binding, is holding ATP in proper conformation through electrostatic interactions. Analysis of the structural change along with calculated reaction pathway suggests that catalytic reaction of phosphotransfer process is a dissociative mechanism^[6]. In addition, theoretical calculations and experiments suggest that a phosphatase reaction follows a general acid–base catalysis associative mechanism in a stepwise mode^[7]. It is expected that more research will be done to gain more insights.

Relevance

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