Investigation of the active site and the conformational stability of nucleoside diphosphate kinase by site-directed mutagenesis.

A D Tepper,H Dammann,A A Bominaar,M Véron

doi:10.1016/s0021-9258(18)31617-x

Abstract

Nucleoside-diphosphate kinase (EC 2.7.4.6) catalyzes phosphate exchange between nucleoside triphosphates and nucleoside diphosphates. Its 17 kDa subunits are highly conserved throughout evolution in both sequence and tertiary structure. Using site-directed mutagenesis we investigated the function of 8 amino acids (Lys16, Tyr56, Arg92, Thr98, Arg109, Asn119, Ser124, and Glu133) that are totally conserved among all nucleoside diphosphate kinases known to date. The mutant proteins all show decreased specific activity and support roles for these residues in catalysis, substrate binding, or both, as was previously proposed on the basis of the x-ray structure (Moréra, S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Véron, M., and Janin, J. (1994) Biochemistry 33, 459-467). Furthermore, residues Lys16, Arg109, and Asn 119 were identified to play important roles in conformational stability or subunit interactions. We show that Lys16 and Asn119 form a rigid structure that is important for enzymatic function and that Arg109, known to interact with the phosphate moiety of the substrate, also plays an important role in subunit association. The dual roles of Lys16, Arg109, and Asn119 in both substrate binding and subunit assembly provide further evidence for a functional coupling between catalytic activity and quaternary structure in nucleoside diphosphate kinase.

Highlights

(Lysis, Wak, g z,ThP, ArglOg,Am"', Ser", and Glu13') The interest in NDP kinhaasserecently been renewed by its that are totally conserved among all nucleoside diphos-involvement in processes not obviously related to the"housephate kinasesk n o w n to date
No structure is available yetfor the human NDP kinases, bugt,iven their high sequence conservation and the ftahcatt they formhexamers
Since the mutant NDP kinases were produced as recombispecific activity, whereas only some of t h r m had altrrrd nant proteins in bacteriiat, was necessary to ascertain that thethermal stahility

Summary

RESULTS AND DISCUSSION

Since the mutant NDP kinases were produced as recombispecific activity, whereas only some of t h r m had altrrrd nant proteins in bacteriiat, was necessary to ascertain that thethermal stahility. Dict.vostelium NDP kinase preparations were not contaminatedoesnotestahlishwhether a crrtain rrsiduc is involvrtl in by the endogenous E. coli enzyme This was im- catalysis or substrate binding. Side chain of NDP kinase Asnllg cannot be replaced by a carboxylate group, as is shown by mutant N119D (0.6% activity). The guanidinium group of Ar$' interacts with the P-phosphate and most likely withthe y-phosphate as it is transferred It should be important both for substrate binding and for catalysis. 0.5%activity, but is presumably retained in thRe109K mutant, which has 5% activity This 10-fold difference between the alanine and lysine substitutions was observed with Arg'', indicating a similar role for residues 92 and 109 in neutralizing the phosphate charges in the active site.The 2-fold higher activity of the Ar$' mutants as compared with that of the. An explanation may lie in the contribution of Mg2' bridging the a- and FIG5..The preformedtemplate in the NDP kinase active site

Schematicrepresentation of the interactions between

WT NDPK OvalbMuymoignlobin

CONCLUSION