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The crystal structure of holo D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extreme thermophile Thermus aquaticus has been solved at 2.5 Angstroms resolution. To study the determinants of thermostability, we compare our structure to four other GAPDHs. Salt links, hydrogen bonds, buried surface area, packing density, surface to volume ratio, and stabilization of alpha-helices and beta-turns are analyzed. We find a strong correlation between thermostability and the number of hydrogen bonds between charged side chains and neutral partners. These charged-neutral hydrogen bonds provide electrostatic stabilization without the heavy desolvation penalty of salt links. The stability of thermophilic GAPDHs is also correlated with the number of intrasubunit salt links and total hydrogen bonds. Charged residues, therefore, play a dual role in stabilization by participating not only in salt links but also in hydrogen bonds with a neutral partner. Hydrophobic effects allow for discrimination between thermophiles and psychrophiles, but not within the GAPDH thermophiles. There is, however, an association between thermostability and decreasing enzyme surface to volume ratio. Finally, we describe several interactions present in both our GAPDH and a hyperthermophilic GAPDH that are absent in the less thermostable GAPDHs. These include a four-residue salt link network, a hydrogen bond near the active site, an intersubunit salt link, and several buried Ile residues.

Our fax numbers: 617-739-9864 and 617-734-4457Our e-mail address: letters@nejm.orgWe cannot acknowledge receipt of your letter, but we will notify you when we have made a decision about publication. We are unable to provide prepublication proofs. Financial associations or other possible conflicts of interest must be disclosed. Submission of a letter constitutes permission for the Massachusetts Medical Society, its licensees, and its assignees to use it in the Journal' s various print and electronic publications and in collections, revisions, and any other form or medium. 1 report that adjuvant chemotherapy with fluorouracil is as effective in older patients with resected colon cancer as it is in younger patients. Their conclusion is based on the absence of a statistically significant interaction between age and treatment effect for both overall and disease-free survival. However, the survival curves for treated and untreated patients who were more than 70 years old seem closer than the curves for treated and untreated younger patients. In our opinion, the absolute gain in survival is the measure that should be discussed with patients when deciding whether to use an adjuvant treatment. We suspect that the absolute differences in the study by Sargent et al. were small, and we would like to know what the absolute gain in survival was at each year of follow-up. 1. Nauta R, Stablein DM, Holyoke ED. Survival of patients with stage B2 colon carcinoma: the Gastrointestinal Tumor Study Group experience. Arch Surg 1989;124:180-2. 2. Gastrointestinal Tumor Study Group. Adjuvant therapy for colon cancerresults of a prospectively randomized trial. N Engl J Med 1984;310:737-43.To the Editor: Sargent et al. state that elderly persons receive adjuvant chemotherapy less frequently than younger patients, citing rates of chemotherapy use from a study that linked 1992 data from the Surveillance, Epidemiology, and End Results (SEER) Program with Medicare data for 1992. 1 However, two more recent studies of SEER-Medicare data report rates of use of adjuvant chemotherapy among elderly persons with stage III colon cancer that are considerably higher than the rates reported by Sargent et al. 2,3 With the use of data from 1992 to 1996, these studies showed that the rate of adjuvant-chemotherapy use wasThe New England Journal of Medicine Downloaded from nejm.org at STOCKHOLMS UNIVERSTITETSBIBL on August 10, 2015. For personal use only. No other uses without permission.

We report the crystal structure of alanine racemase from Mycobacterium tuberculosis (Alr(Mtb)) at 1.9 A resolution. In our structure, Alr(Mtb) is found to be a dimer formed by two crystallographically different monomers, each comprising 384 residues. The domain makeup of each monomer is similar to that of Bacillus and Pseudomonas alanine racemases and includes both an alpha/beta-barrel at the N-terminus and a C-terminus primarily made of beta-strands. The hinge angle between these two domains is unique for Alr(Mtb), but the active site geometry is conserved. In Alr(Mtb), the PLP cofactor is covalently bound to the protein via an internal aldimine bond with Lys42. No guest substrate is noted in its active site, although some residual electron density is observed in the enzyme's active site pocket. Analysis of the active site pocket, in the context of other known alanine racemases, allows us to propose the inclusion of conserved residues found at the entrance to the binding pocket as additional targets in ongoing structure-aided drug design efforts. Also, as observed in other alanine racemase structures, PLP adopts a conformation that significantly distorts the planarity of the extended conjugated system between the PLP ring and the internal aldimine bond.

Based on crystal structure analysis of the Serratia nuclease and a sequence alignment of six related nucleases, conserved amino acid residues that are located in proximity to the previously identified catalytic site residue His89 were selected for a mutagenesis study. Five out of 12 amino acid residues analyzed turned out to be of particular importance for the catalytic activity of the enzyme: Arg57, Arg87, His89, Asn119 and Glu127. Their replacement by alanine, for example, resulted in mutant proteins of very low activity, < 1% of the activity of the wild-type enzyme. Steady-state kinetic analysis of the mutant proteins demonstrates that some of these mutants are predominantly affected in their kcat, others in their Km. These results and the determination of the pH and metal ion dependence of selected mutant proteins were used for a tentative assignment for the function of these amino acid residues in the mechanism of phosphodiester bond cleavage by the Serratia nuclease.

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In an x-ray diffraction study by the isomorphous replacement method, the structure of the complex of aspartate carbamoyltransferase (EC 2.1.3.2) bound to the bisubstrate analogue N-(phosphonacetyl)-L-aspartate has been solved to 2.9-A resolution (R = 0.24). The large quaternary structural changes previously deduced by molecular replacement methods have been confirmed: the two catalytic trimers (c3) move apart by 12 A and mutually reorient by 10°, and the regulatory dimers (r2) reorient each about its twofold axis by about 15°. In this, the T-to-R transition, new 3). The structure consists of catalytic (c) and regulatory (r) chains in a (c3)2(r2)3 oligomer which has D3 symmetry ( Fig. 1) (4-6). Previous x-ray diffraction studies have established the structure of the less active T form to a resolution of 2.6 A (7,8). Also, the large changes in quaternary structure that occur in the transition to the R form as the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA) is bound have been described (9). Less precise indications of these large conformational changes have been established by other investigators (10-13). Our refinement of this R form proceeded so slowly that we have undertaken an independent solution of the R structure bound to PALA by the method of multiple isomorphous replacement (MIR). The results, described below, establish the binding site of PALA and elucidate previously undescribed changes in tertiary structure of the catalytic chains, and they also enable us to look C6 FIG. 1. Subunit and domain structure of aspartate carbamoyltransferase, as viewed down the threefold axis. Domains of the catalytic (c) chains C1-C6 are equatorial (eq) and polar (po), while those of the regulatory (r) chains R1-R6 are zinc (zn) and allosteric (al).at the T-to-R homotropic transition as typified by these two x-ray structures: unligated-and PALA-aspartate carbamoyltransferase. MATERIALS AND METHODSCrystal Growth. The enzyme was isolated and purified (14) from regulatory mutant E. coli provided by J. C. Gerhart and grown at the New England Enzyme Center. Crystals were obtained by dialyzing for 1-2 weeks at 21°C against 50 mM maleic acid/1 mM PALA/3 mM NaN3/N-ethylmorpholine to pH 5.9 and conductivity 3.4 inS. Temperature control was critical for crystallization. After growth, the crystals were transferred, still within their microdialysis vials, to 20% (wt/vol) polyethylene glycol-6000/crystallization buffer at pH 5.8. 1643The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.