Baker's Yeast Adenlate Kinase

Abstract

(ATPPS) were used to determine the stereochemical mode of coordination of Mg2+ to ATP when bound in the yeast adenylate kinase complex. ATPaS isomer A is the preferred substrate either in the presence of Mg" or Cd2+ ; ratios of the rate of reaction at infinite concentration of substrate, V, for A to B isomers of ATPclS with MgZ + were 457/51.4 = 8.9 and with CdZC 124/12.8 = 9.7. Thus there is no reversal of stereospecificity on substituting Cd2+ for Mg2+ and this supports the view that metal does not chelate with the a-P. In contrast the V ratios of ATPPS(A) to ATPPS(B) withMg2+ were 1.33/0.15 = 8.9 andwithCd" O.lSj5.l = 0.035.Thusthisreversal ofstereospecificitywhenCd'+ is substituted for MgL + (in view of the fact that Mg2 + binds to 0 and Cd2+ to S) leads to the conclusion that a metal chelation must occur via the p-phosphate. Since the enzyme prefers MgATPPS(A) and CdATPPS(B) (both isomers have A-screw sense) to MgATPpS(B) and CdATPPS(A) (both isomers have A-screw sense) and Mg2+ also coordinates with the y-phosphate (by analogy with other adenylate kinase isozymes), the interpretation is that baker' yeast adenylate kinase binds MgATP as A , P, y. The fluorescence of the two Trp residues of yeast adenylate kinase has been used to investigate conformational changes of the enzyme on binding its substrates. Fluorescence is maximal at 334 nm (A,,, 295 nm, pH 7.5) and is reduced 10 % with no shift in A, , , in the presence of ATP and reduced a further 15 % on the addition of Mg2+ or other metal acitvators to E-ATP. In contrast non-activating metals such as Ba2+ and Sr2+ increase quenching only slightly. With ITP, UTP, CTP, GTP the quenching is 4.1 %, 5.2 %, 8.7 % and 6.6 % with or without MgL +. Differential conformational changes ofyeast adenylate kinase on binding MgATP, ATP, and other nucleotides are shown by (a) fluorescence changes, (b) previously reported kinetic and binding studies and (c) by the fact that the K , for AMP in the presence of MgATP is 17 times less than in the presence of MgGTP. These differential conformational changes result in differing tendencies of AMP to bind to the enzyme. Ultraviolet difference spectra obtained on binding of ATP to the enzyme (shoulder at 271 nm and peak at 276 nni) have been compared with the difference spectra of the interaction of ATP, adenosine and I-methyl adenosine respectively with methyl mercuric hydroxide and formaldehyde. On the basis of these comparisons, the shoulder at 271 nm reflects the interaction of N(l) and the peak at 276 nm the interaction of the 6-amino group of ATP with the enyzme.The following mechanism of reaction is proposed : upon binding MgATP the enzyme undergoes a conformational change which closes the active-site crevice by bringing the two lobes closer, expelling water from the crevice. AMP is bound with higher affinity and the a-oxygen attacks the y-phosphate of ATP in a nucleophilic reaction. correct and complete conformational change necessary for an appropriate binding of AMP at the AMP site and to give maximal activity; (c) nucleot...

Structural Investigations of the Mg · ATP Complex at the Active Site of Porcine Adenylate Kinase Using Phosphorothioate Analogs and Electron Paramagnetic Resonance of Mn(II) with Chiral 17)‐Labelled ATP Analogs

“…A further difference in the behaviour of the two enzymes was noted in their activity with ATP[:/S]. As reported in [25] and confirmed by us in these studies, adenylate kinase from baker's yeast is a factor of about 4-6 less active with both ATP and ATP[/S] in the presence of Cd2+ when compared with the activity in the presence of Mg2+. Although a similar Factor is found for ATP and porcine adenylate kinase, ATP[/S] reacts 160-fold more slowly with Cd2+ as activating ion than with Mg2+.…”

ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites

“…We speculate that AMP might bind the ATP-binding site under these conditions, which may increase *AMP labeling at the AMP-binding site(s). This speculation is founded on results with bakers' yeast and porcine muscle adenylate kinase showing that ( a ) ATP facilitated AMP binding, and ( b ) in the absence of ATP, AMP bound preferentially to the ATP-binding site although with much lower affinity than ATP (32, 33, 58). …”

Domain movement in rabbit muscle adenylate kinase might involve proline isomerization

“…X‐ray analysis of enzyme crystals revealed that the AMP‐binding domain undergoes a movement of 8 Å upon AMP binding and that the ATP‐binding domain moves up to 30 Å upon binding with ATP [7, 8]. This was strongly supported by investigations of different ligand forms of AK in solution using several methods [9–16]. However, these and other published investigations have paid little attention to whether these substrate‐induced conformational changes involve proline isomerization.…”