Substitution of a serine residue for proline-87 reduces catalytic activity and increases susceptibility to proteolysis of Escherichia coli adenylate kinase.

Abstract

Amino acid analysis, HPLC separation of trypsin digests, and sequence analysis showed that the thermosensitivity of the adenylate kinase (EC 2.7.4.3) from Escherichia coli K-12 strain CR341 T28 results from substitution of a serine residue for proline-87 in the wild-type enzyme. This mutation is accompanied by decreased affinity for nucleotide substrates and decreased catalysis. Circular dichroism spectroscopy showed a significant change of the secondary structure. This mainly corresponds to a reduction in a-helix content (39%) of mutant protein as compared to wild-type adenylate kinase (50%). Altered conformation of thermosensitive adenylate kinase was also manifested by an increase in susceptibility to proteolysis by trypsin. Ap5A and ATP, known to induce important conformational changes in eukaryotic adenylate kinase(s), protected the mutant enzyme against inactivation by trypsin. This seems to indicate that the "loosening" of the three-dimensional structure of E. coli adenylate kinase by proline -+ serine substitution is largely compensated for when an enzyme-ATP or enzyme-Ap5A complex is formed.Adenylate kinase (EC 2.7.4.3) is an essential enzyme that regenerates ADP from ATP and AMP by the reaction MgATP + AMP -MgADP + ADP. The relatively small size of the enzyme makes it a very interesting model for structural studies. During the last decade, muscle adenylate kinase (myokinase) has been purified from man, pig, rabbit, and calf, crystallized, and investigated extensively by various physicochemical approaches (1-6).Isolation and genetic characterization of thermosensitive mutants of Escherichia coli exhibiting a complex phenotype (defective synthesis of phospholipids, RNA, and ATP at nonpermissive temperatures) allowed localization of mutation to the adk gene (7-10). This opened the way for molecular cloning of the adk gene, deduction of its primary structure from the nucleotide sequence (11), and overproduction of the protein.Thermosensitive adenylate kinase of E. coli has the same molecular weight as wild-type enzyme but a lower affinity for nucleotides and a Vma, value that is only 10-15% that of the parental enzyme (12)(13)(14). Thermosensitive adenylate kinase is irreversibly inactivated by incubation of crude extracts at 40°C (7,(15)(16)(17) due to proteolysis subsequent to thermal denaturation (or transconformation) of mutant enzyme at this temperature (17). It is therefore important to locate the site of mutation to gain a better understanding of the structural factors involved in the maintenance of a stable catalytically active conformation of bacterial adenylate kinase.In this paper, we show that thermosensitive adenylate kinase differs from wild-type in the substitution of a serine residue for a proline residue at position 87. Some consequences of this mutation for the secondary and tertiary structure of adenylate kinase have been investigated by circular dichroism (CD) spectroscopy as well as by susceptibility to proteolysis by trypsin. MATERIALS AND METHODSChemicals. Adenine nucleotides...

Direct Mg2+ Binding Activates Adenylate Kinase from Escherichia coli

“…6. Asp-84 is very close to the two residues, Phe-86 and Pro-87, which were previously shown to abolish the sigmoidal AMP inhibition behavior discussed above (34,35). This physical picture is also consistent with the proposed second-order correction to the model where Mg 2ϩ can exchange binding sites in the highly flexible interior of the enzyme (cf.…”

“…For example, one of the most puzzling discrepancies is the change in turnover rates with increasing AMP concentration between rabbit muscle AK and Escherichia coli AK. Although the reactivity of rabbit muscle AK is slightly inhibited at higher AMP concentrations (29, 32), E. coli AK exhibits its maximum turnover rate around 0.2 mM AMP followed by a steep drop, which plateaus at still higher AMP concentrations (33)(34)(35). This observation has been traditionally attributed to greater substrate inhibition by AMP in E. coli AK compared with the rabbit isoform; yet, the issue of whether the reaction involves competitive or non-competitive inhibition by AMP at the ATP binding site remains unresolved (15,33,(35)(36)(37).…”

Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria

“…This represents the phosphate binding loop characteristic of all AK. Glaser et al [19,35] have proposed that the sequence -G-F-P-R-(Gly84-Phe85-Pro86-Arg87) present in the core domain plays a crucial role in stabilizing the tertiary structure of the enzyme. In all our three structures, Pro86 adopts a cis conformation similarly to the other structurally characterized AK.…”

Stability, Activity and Structure of Adenylate Kinase Mutants

“…The observed mutation effects could be reasonably interpreted in view of the spatial structure (Muller and Schulz, 1992). Some of the work included the analysis of thermodynamic stabilities (Gilles et al, 1986;Tian et al, 1990;Liang et al, 1991 ;Okajima et al, 1991), but no helix capping or core perturbation.…”