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J. Biol. Chem., Vol. 277, Issue 8, 5749-5755, February 22, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/8/5749    most recent M106538200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kasrayan, A. Articles by Sjöberg, B.-M. Search for Related Content PubMed PubMed Citation Articles by Kasrayan, A. Articles by Sjöberg, B.-M. Social Bookmarking                      What's this?

The Conserved Active Site Asparagine in Class I Ribonucleotide Reductase Is Essential for Catalysis^*

Alex Kasrayan, Annika L. Persson, Margareta Sahlin, and Britt-Marie Sjöberg^§

From the Department of Molecular Biology and Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden

The active site residue Asn-437 in protein R1 of the Escherichia coli ribonucleotide reductase makes a hydrogen bond to the 2'-OH group of the substrate. To elucidate its role(s) during catalysis, Asn-437 was engineered by site-directed mutagenesis to several other side chains (Ala, Ser, Asp, Gln). All mutant proteins were incapable of enzymatic turnover but promoted rapid protein R2 tyrosyl radical decay in the presence of the k_cat inhibitor 2'-azido-2'-deoxy-CDP with similar decay rate constants as the wild-type R1. These results show that all Asn-437 mutants can perform 3'-H abstraction, the first substrate-related step in the reaction mechanism. The most interesting observation was that three of the mutant proteins (N437A/S/D) behaved as suicidal enzymes by catalyzing a rapid tyrosyl radical decay also in reaction mixtures containing the natural substrate CDP. The suicidal CDP-dependent reaction was interpreted to suggest elimination of the substrate's protonated 2'-OH group in the form of water, a step that has been proposed to drive the 3'-H abstraction step. A furanone-related chromophore was formed in the N437D reaction, which is indicative of stalling of the reaction mechanism at the reduction step. We conclude that Asn-437 is essential for catalysis but not for 3'-H abstraction. We propose that the suicidal N437A, N437S, and N437D mutants can also catalyze the water elimination step, whereas the inert N437Q mutant cannot. Our results suggest that Asn-437, apart from hydrogen bonding to the substrate, also participates in the reduction steps of catalysis by class I ribonucleotide reductase.

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