Mechanism of adenylate kinase. The conserved aspartates 140 and 141 are important for transition state stabilization instead of substrate-induced conformational changes.

Dahnke, Terri; Tsai, Ming‐Daw

doi:10.1016/s0021-9258(17)37162-4

Cited by 16 publications

(7 citation statements)

References 24 publications

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“…, k cat and K M ; Table ). As expected, the mutation substantially reduced the catalytic activity of the enzyme (58-fold in k cat ), while the change in K M is relatively small, and a similar level of change in k cat and K M was also reported for chicken muscle AdK . This result suggests its involvement only in the catalytic reaction and thus corroborates our prediction from MD simulations.…”

Section: Resultssupporting

confidence: 90%

Mechanistic Basis for a Connection between the Catalytic Step and Slow Opening Dynamics of Adenylate Kinase

Dulko-Smith

Ojeda-May

Ådén

et al. 2023

J. Chem. Inf. Model.

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Escherichia coli adenylate kinase (AdK) is a small, monomeric enzyme that synchronizes the catalytic step with the enzyme's conformational dynamics to optimize a phosphoryl transfer reaction and the subsequent release of the product. Guided by experimental measurements of low catalytic activity in seven single-point mutation AdK variants (K13Q, R36A, R88A, R123A, R156K, R167A, and D158A), we utilized classical mechanical simulations to probe mutant dynamics linked to product release, and quantum mechanical and molecular mechanical calculations to compute a free energy barrier for the catalytic event. The goal was to establish a mechanistic connection between the two activities. Our calculations of the free energy barriers in AdK variants were in line with those from experiments, and conformational dynamics consistently demonstrated an enhanced tendency toward enzyme opening. This indicates that the catalytic residues in the wild-type AdK serve a dual role in this enzyme's function�one to lower the energy barrier for the phosphoryl transfer reaction and another to delay enzyme opening, maintaining it in a catalytically active, closed conformation for long enough to enable the subsequent chemical step. Our study also discovers that while each catalytic residue individually contributes to facilitating the catalysis, R36, R123, R156, R167, and D158 are organized in a tightly coordinated interaction network and collectively modulate AdK's conformational transitions. Unlike the existing notion of product release being rate-limiting, our results suggest a mechanistic interconnection between the chemical step and the enzyme's conformational dynamics acting as the bottleneck of the catalytic process. Our results also suggest that the enzyme's active site has evolved to optimize the chemical reaction step while slowing down the overall opening dynamics of the enzyme.

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Section: Resultssupporting

confidence: 90%

Mechanistic Basis for a Connection between the Catalytic Step and Slow Opening Dynamics of Adenylate Kinase

Dulko-Smith

Ojeda-May

Ådén

et al. 2023

J. Chem. Inf. Model.

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“…This residue was shown to be a key catalytic residue in AK1 chick (R138) and its function could not be replaced by the positively charged lysine (Yan et al, 1990). Two aspartate residues, namely D140 and D141 of AK1 chick (D139 and D140 of UK dicty ) that are also part of the LID domain are believed to be important for transition state stabilization but not for substrate-induced conformational changes (Dahnke & Tsai, 1994). According to our structure, this would leave R131 as the prime candidate to be the trigger that causes closure of the LID domain over bound substrate since it interacts with oxygens of PR and Pγ of the ATP binding site.…”

Section: Resultsmentioning

confidence: 87%

Crystal Structure of the Complex of UMP/CMP Kinase from Dictyostelium discoideum and the Bisubstrate Inhibitor P¹-(5‘-Adenosyl) P⁵-(5‘-Uridyl) Pentaphosphate (UP₅A) and Mg²⁺ at 2.2 Å: Implications for Water-Mediated Specificity

et al. 1996

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The three-dimensional structure of the UMP/CMP kinase (UK) from the slime mold Dictyostelium discoideum complexed with the specific and asymmetric bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-uridyl) pentaphosphate (UP5A) has been determined at a resolution of 2.2 A. The structure of the enzyme, which has up to 41% sequence homology with known adenylate kinases (AK), represents a closed conformation with the flexible monophosphate binding domain (NMP site) being closed over the uridyl moiety of the dinucleotide. Two water molecules were found within hydrogen-bonding distance to the uracil base. The key residue for the positioning and stabilization of those water molecules appears to be asparagine 97, a residue that is highly specific for AK-homologous UMP kinases, but is almost invariably a glutamine in adenylate kinases. Other residues in this region are highly conserved among AK-related NMP kinases. The catalytic Mg2+ ion is coordinated with octahedral geometry to four water molecules and two oxygens of the phosphate chain of UP5A but has no direct interactions with the protein. The comparison of the geometry of the UKdicty.UP5A.Mg2+ complex with the previously reported structure of the UKyeast.ADP.ADP complex [Müller-Dieckmann & Schulz (1994) J. Mol. Biol. 236, 361-367] suggests that UP5A in our structure mimics an ADP.Mg.UDP biproduct inhibitor rather than an ATP. MG.UMP bisubstrate inhibitor.

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“…The importance of protein interactions with the tripolyphosphoryl moiety of ATP (or GTP) for the stabilization of the transition state seems to be a rather universal property of phosphortransferases. There are numerous examples in the literature where mutation of residues involved in such interactions produce significant changes in k cat with little or no change in K m : adenylate kinase (Arg132Met) (Dahnke & Tsai, 1994), adenylosuccinate synthetase (Gly15Val) (Kang & Fromm, 1994), the 70-kDa heat shock cognate protein (Asp199Ser) (Wilbands et al, 1994), and Rho protein (Asp265Asn) (Dombroski et al, 1988).…”

Section: Purification Of Wild-type and Mutant Human Brainmentioning

confidence: 99%

ATP-Binding Site of Human Brain Hexokinase As Studied by Molecular Modeling and Site-Directed Mutagenesis

et al. 1996

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The interaction of ATP with the active site of hexokinase is unknown since the crystal structure of the hexokinase-ATP complex is unavailable. It was found that the ATP binding site of brain hexokinase is homologous to that of actin, heat shock protein hsc70, and glycerol kinase. On the basis of these similarities, the ATP molecule was positioned in the catalytic domain of human brain hexokinase, which was modeled from the X-ray structure of yeast hexokinase. Site-directed mutagenesis was performed to test the function of residues presumably involved in interaction with the tripolyphosphoryl moiety of ATP. Asp532, which is though to be involved in binding the Mg2+ ion of the MgATP2- complex, was mutated to Lys and Glu. The kcat values decreased 1000- and 200-fold, respectively, for the two mutants. Another residue, Thr680 was proposed to interact with the gamma-phosphoryl group of ATP through hydrogen bonds and was mutated to Val and Ser. The kcat value of the Thr680Val mutant decreased 2000-fold, whereas the kcat value of the Thr680Ser decreased only 2.5-fold, implying the importance of the hydroxyl group. The Km and dissociation constant values for either ATP or glucose of all the above mutants showed little or no change relative to the wild-type enzyme. The Ki values for the glucose 6-phosphate analogue 1,5-anhydroglucitol 6-phosphate, were the same as that of the wild-type enzyme, and the inhibition was reversed by inorganic phosphate (Pi) for all four mutants. The circular dichroism spectra of the mutants were the same as that of the wild-type enzyme. The results from the site-directed mutagenesis demonstrate that the presumed interactions of investigated residues with ATP are important for the stabilization of the transition state.

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Mechanism of adenylate kinase. The conserved aspartates 140 and 141 are important for transition state stabilization instead of substrate-induced conformational changes.

Cited by 16 publications

References 24 publications

Mechanistic Basis for a Connection between the Catalytic Step and Slow Opening Dynamics of Adenylate Kinase

Mechanistic Basis for a Connection between the Catalytic Step and Slow Opening Dynamics of Adenylate Kinase

Crystal Structure of the Complex of UMP/CMP Kinase from Dictyostelium discoideum and the Bisubstrate Inhibitor P¹-(5‘-Adenosyl) P⁵-(5‘-Uridyl) Pentaphosphate (UP₅A) and Mg²⁺ at 2.2 Å: Implications for Water-Mediated Specificity

ATP-Binding Site of Human Brain Hexokinase As Studied by Molecular Modeling and Site-Directed Mutagenesis

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Mechanism of adenylate kinase. The conserved aspartates 140 and 141 are important for transition state stabilization instead of substrate-induced conformational changes.

Cited by 16 publications

References 24 publications

Mechanistic Basis for a Connection between the Catalytic Step and Slow Opening Dynamics of Adenylate Kinase

Mechanistic Basis for a Connection between the Catalytic Step and Slow Opening Dynamics of Adenylate Kinase

Crystal Structure of the Complex of UMP/CMP Kinase from Dictyostelium discoideum and the Bisubstrate Inhibitor P1-(5‘-Adenosyl) P5-(5‘-Uridyl) Pentaphosphate (UP5A) and Mg2+ at 2.2 Å: Implications for Water-Mediated Specificity

ATP-Binding Site of Human Brain Hexokinase As Studied by Molecular Modeling and Site-Directed Mutagenesis

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Resources

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Crystal Structure of the Complex of UMP/CMP Kinase from Dictyostelium discoideum and the Bisubstrate Inhibitor P¹-(5‘-Adenosyl) P⁵-(5‘-Uridyl) Pentaphosphate (UP₅A) and Mg²⁺ at 2.2 Å: Implications for Water-Mediated Specificity