Structural and functional consequences of amino acid substitutions in the second conserved loop of Escherichia coli adenylate kinase.


Abstract

All known nucleoside monophosphate kinases contain an invariant sequence Asp-Gly-Phe(Tyr)-Pro-Arg. In order to understand better the structural and functional role of individual amino acid residues belonging to the above sequence, three mutants of Escherichia coli adenylate kinase (D84H, G85V, and F86L) were produced by site-directed mutagenesis. Circular dichroism spectra revealed that the secondary structure dichroism spectra revealed that the secondary structure of all three mutant proteins is very similar to that of the wild-type enzyme. However, each of the substitutions resulted in a decreased thermodynamic stability of the protein, as indicated by differential scanning calorimetry measurements and equilibrium unfolding experiments in guanidine HCl. The destabilizing effect was most pronounced for the G85V mutant, in which case the denaturation temperature was decreased by as much as 11 degrees C. The catalytic activity of the three mutants represented less than 1% of that of the wild-type enzyme. Furthermore, for the D84H-modified form of adenylate kinase, the impaired binding of nucleotide substrates was accompanied by a markedly decreased affinity for magnesium ion. These observations support the notion that Asp84 is directly involved in binding of nucleotide substrates and that this binding is mediated by interaction of the aspartic acid residue with divalent cation. The two remaining residues probed in this study, Gly85 and Phe86, belong to a beta-turn which appears to play a major role in stabilizing the three-dimensional structure of adenylate kinase. Study holds ProTherm entries: 98, 99, 100, 101, 13184, 13185, 13186 Extra Details: Escherichia coli adenylate kinase; thermodynamic stability;,differential scanning calorimetry; three-dimensional structure

Submission Details

ID: ErZCTQaw

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:14 p.m.

Version: 1

Publication Details
Rose T;Glaser P;Surewicz WK;Mantsch HH;Reinstein J;Le Blay K;Gilles AM;Bârzu O,J. Biol. Chem. (1991) Structural and functional consequences of amino acid substitutions in the second conserved loop of Escherichia coli adenylate kinase. PMID:1748642
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
6F7U 2018-03-14 1.4 Molecular Mechanism of ATP versus GTP Selectivity of Adenylate Kinase
4X8L 2015-07-22 1.7 Crystal structure of E. coli Adenylate kinase P177A mutant in complex with inhibitor Ap5a
1E4Y 2000-08-04 1.85 Mutant P9L of adenylate kinase from E. coli, modified in the Gly-loop
1E4V 2000-08-04 1.85 Mutant G10V of adenylate kinase from E. coli, modified in the Gly-loop
5EJE 2016-11-09 1.9 Crystal structure of E. coli Adenylate kinase G56C/T163C double mutant in complex with Ap5a
1AKE 1994-01-31 2.0 STRUCTURE OF THE COMPLEX BETWEEN ADENYLATE KINASE FROM ESCHERICHIA COLI AND THE INHIBITOR AP5A REFINED AT 1.9 ANGSTROMS RESOLUTION: A MODEL FOR A CATALYTIC TRANSITION STATE
3HPR 2009-11-03 2.0 Crystal structure of V148G adenylate kinase from E. coli, in complex with Ap5A
1ANK 1994-05-31 2.0 THE CLOSED CONFORMATION OF A HIGHLY FLEXIBLE PROTEIN: THE STRUCTURE OF E. COLI ADENYLATE KINASE WITH BOUND AMP AND AMPPNP
3HPQ 2009-11-03 2.0 Crystal structure of wild-type adenylate kinase from E. coli, in complex with Ap5A
4X8O 2015-07-15 2.1 Crystal structure of E. coli Adenylate kinase Y171W mutant in complex with inhibitor Ap5a
4AKE 1996-06-10 2.2 ADENYLATE KINASE
4X8H 2015-07-22 2.5 Crystal structure of E. coli Adenylate kinase P177A mutant
4X8M 2015-07-15 2.6 Crystal structure of E. coli Adenylate kinase Y171W mutant
2ECK 1997-03-12 2.8 STRUCTURE OF PHOSPHOTRANSFERASE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.1 Adenylate kinase A8GAV3 KAD_SERP5
95.8 Adenylate kinase A8AJW9 KAD_CITK8
96.3 Adenylate kinase P0A1V4 KAD_SALTY
96.3 Adenylate kinase P0A1V5 KAD_SALTI
96.3 Adenylate kinase B4TMG6 KAD_SALSV
96.3 Adenylate kinase B5BD44 KAD_SALPK
96.3 Adenylate kinase C0Q812 KAD_SALPC
96.3 Adenylate kinase Q5PFK8 KAD_SALPA
96.3 Adenylate kinase B4SWY1 KAD_SALNS
96.3 Adenylate kinase B4T9I1 KAD_SALHS
96.3 Adenylate kinase B5R612 KAD_SALG2
96.3 Adenylate kinase B5QU77 KAD_SALEP
96.3 Adenylate kinase B5FLJ7 KAD_SALDC
96.3 Adenylate kinase Q57S76 KAD_SALCH
96.3 Adenylate kinase B5EXN0 KAD_SALA4
95.8 Adenylate kinase A4W7F8 KAD_ENT38
96.3 Adenylate kinase A6T5N7 KAD_KLEP7
96.3 Adenylate kinase B5Y0N3 KAD_KLEP3
99.1 Adenylate kinase B7MDZ6 KAD_ECO45
99.1 Adenylate kinase Q3Z4S5 KAD_SHISS
99.5 Adenylate kinase Q83M40 KAD_SHIFL
99.5 Adenylate kinase Q8FK84 KAD_ECOL6
99.5 Adenylate kinase Q0TKG7 KAD_ECOL5
99.5 Adenylate kinase B7MQI9 KAD_ECO81
99.5 Adenylate kinase B7NIF6 KAD_ECO7I
99.5 Adenylate kinase B7UKF4 KAD_ECO27
99.5 Adenylate kinase Q32J54 KAD_SHIDS
99.5 Adenylate kinase B7M3W6 KAD_ECO8A
100.0 Adenylate kinase Q0T7B1 KAD_SHIF8
100.0 Adenylate kinase Q325C2 KAD_SHIBS
100.0 Adenylate kinase B2U4S7 KAD_SHIB3
100.0 Adenylate kinase B7LV13 KAD_ESCF3
100.0 Adenylate kinase B1LJN2 KAD_ECOSM
100.0 Adenylate kinase B6I0C6 KAD_ECOSE
100.0 Adenylate kinase B7N927 KAD_ECOLU
100.0 Adenylate kinase P69441 KAD_ECOLI
100.0 Adenylate kinase B1IZC0 KAD_ECOLC
100.0 Adenylate kinase A7ZXD2 KAD_ECOHS
100.0 Adenylate kinase B1XFR1 KAD_ECODH
100.0 Adenylate kinase C4ZUS8 KAD_ECOBW
100.0 Adenylate kinase P69442 KAD_ECO57
100.0 Adenylate kinase B7L799 KAD_ECO55
100.0 Adenylate kinase A7ZIN4 KAD_ECO24