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letters: Mary A. Dwyer, Loren L. Looger, and Homme W. Hellinga Retraction Science 2008; 319: 569b [Full text] [PDF]

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Comments on M. A. Dwyer et al. Retraction

Jack F. Kirsch   (10 March 2008)

Comment toward M. A. Dwyer et al. Retraction

John Richard   (10 March 2008)

Comments on M. A. Dwyer et al. Retraction 10 March 2008
Jack F. Kirsch QB3 Institute, University of California, Berkeley, CA 94720–3220, USA Respond to this E-Letter: Re: Comments on M. A. Dwyer et al. Retraction	In my view, the Dwyer et al. Retraction (1 February 2008, p. 569) of their 2004 Report (1) does not explain the published results. They attribute the triosephosphate isomerase (TIM) activity reported in the earlier work solely to contamination by wild-type (TIM). The contamination is certainly real as originally documented by John Richard, but this fact does not explain the reported Km values, which are more than an order of magnitude reduced from that of the wild-type enzyme. While Vmax values can be in error due to poor estimates of enzyme concentration or the presence of inactive enzyme, and Km values may be artificially higher due to the presence of competitive inhibitors, it is hard to explain the much lower values reported for the designed enzyme. The Retraction does not address this issue, which is fundamental. Second, contamination by wild-type enzyme would be expected to compromise randomly (dependent on expression levels) the kinetic parameters of all designed TIM variants, yet Fig. 4C would make sense only if the design were successful. The active TIM design included three active site residues. The reported data show systematic reductions in activity from the complete designed TIM as each of the design entities is replaced by alanine, and the triple alanine replacement is the most compromised of all. Simple contamination with wild-type TIM cannot explain such a result. Additionally the statement, “The in vivo experiments have not been reexamined.” does not address what to many is the most convincing part of the paper—i.e., the in vivo rescue of a TIM knockout in Escherichia coli by the designed NovoTIM. There is no way that wild-type TIM contamination in the in vitro experiments could account for that in vivo result. Jack F. Kirsch QB3 Institute, University of California, Berkeley, CA 94720–3220, USA. Reference 1. M. A. Dwyer, L. L. Looger, H. W. Hellinga, Science 304, 1967 (2004).
Comment toward M. A. Dwyer et al. Retraction 10 March 2008
John Richard Department of Chemistry, The State University of New York, Buffalo, NY 14260–3000, USA Respond to this E-Letter: Re: Comment toward M. A. Dwyer et al. Retraction	I read with interest the Retraction of the Report "Computational design of a biologically active enzyme" (1) that was published on page 569 of your 1 February issue. In this Retraction, the authors mention a "reanalysis" carried out in my laboratory at the University at Buffalo. Reanalysis is an odd way to characterize our routine separation of host wild-type triosephosphate isomerase from an inactive engineered overexpressed protein. This purification was facilitated by the determination that the Michaelis constant Km = 2.3 mM for isomerization of dihydroxyacetone phosphate (DHAP) catalyzed by the active enzyme in our preparation of the designed protein is that expected for wild-type Escherichia coli triosephosphate isomerase. Dr. Hellinga's paper in Science reported a range of values of Km = 0.10 to 0.33 mM for isomerization of DHAP catalyzed by various iterations of his designed proteins (Table 2 of the Report in Science), which are considerably smaller than Km ≈ 2 mM expected for wild-type triosephosphate isomerase from E. coli. In a later report in Journal of Molecular Biology (2) the value of Km = 0.18 mM for the ecNovoTIM1.2 construct was amended, without adequate explanation, to a 40-fold larger value of 7.1 mM (Table 1 of that paper). The problems experienced by members of Dr. Hellinga's laboratory in determining accurate values of Km are closely tied to their failure to identify the isolated enzymatic activity as wild-type E. coli triosephosphate isomerase and to the unfortunate acceptance of the original papers for publication in Science and the Journal of Molecular Biology. In order to minimize the speculation that will arise when these problems become public knowledge, I think that they should have been addressed in the recent Retraction. John Richard Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY 14260–3000, USA. References 1. M. A. Dwyer, L. L. Looger, H. W. Hellinga, Science 304, 1967 (2004). 2. M. Allert, M. A. Dwyer, H. W. Hellinga, J. Mol. Biol. 366, 945 (2007).