Current FDA-approved l-asparaginases also possess significant l-glutaminase activity, which correlates with many of the toxic side effects of these drugs. Therefore, l-asparaginases with reduced l-glutaminase activity are predicted to be safer. We exploited our recently described structures of the Erwinia chrysanthemi l-asparaginase (ErA) to inform the design of mutants with diminished ability to hydrolyze l-glutamine. Structural analysis of these variants provides insight into the molecular basis for the increased l-asparagine specificity. A primary role is attributed to the E63Q mutation that acts to hinder the correct positioning of l-glutamine but not l-asparagine. The substitution of Ser-254 with either an asparagine or a glutamine increases the l-asparagine specificity but only when combined with the E63Q mutation. The A31I mutation reduces the substrate Km value; this is a key property to allow the required therapeutic l-asparagine depletion. Significantly, an ultra-low l-glutaminase ErA variant maintained its cell killing ability. By diminishing the l-glutaminase activity of these highly active l-asparaginases, our engineered ErA variants hold promise as l-asparaginases with fewer side effects.

On the basis of literature data and from our own experiments, the average enthalpies (Δ - H r ) for different types of hydrolytic enzymatic reactions have been found: for peptide bond hydrolysis Δ - H r = −7.6±2.0 kJ mol −1 ; for amide bond hydrolysis Δ- H r = −24.7±2.0 kJ mol −1 ; for ester bond hydrolysis Δ - H r = 1.8 kJ mol −1 . Furthermore, Δ- H r values have been calculated for yarious types of hydrolysis reaction in orthophosphate esters and their derivatives; Δ - H r values for other types of hydrolytic enzymatic reactions are also cited from the literature.

The enthalpy of the asparagine hydrolysis reaction is found to be Δ H 1 = −24.56 ± 0.35 kJ mol −1 (0.05 M phosphate buffer, pH 6.86, 298.15 K); besides, the enthalpies of ionization of asparagine and aspartic acid are also determined Δ H 2 = −39.7 ± 0.3 kJ mol −1 Δ H 3 = −38.6 ± 0.4 kJ mol −1 Δ H 4 = −3.83 ± 0.10kJ mol −1 Using values of the enthalpies Δ H 1 to Δ H 4 , the plot of enthalpy variation of the asparagine hydrolysis reaction versus pH (3–10) is plotted. References 1 C. Kitzinger R. Hems Biochem. J. 71 1955 395 2 T. Benzinger C. Kitzinger Fed. Proc., Fed. Am. Soc. Exp. 13 1954 11 3 T. Benzinger C. Kitzinger Abstr. 10th Int. Physiol. Congr., Brussels 1956 85 4 C. Kitzinger T. Benzinger Z. Naturforsch., Teil B 10 1955 375 5 H.A. Sober Handbook of Biochemistry and Selected Data for Molecular Biology 1968 The Chemical Rubber Co Boca Raton, FL 6 M.V. Rekharsky L.D. Rumsh V.K. Antonov G.L. Galchenko Thermochim. Acta 81 1984 167 7 M.V. Rekharsky G.L. Galchenko A.M. Egorov Zh. Obshch. Khim. 54 1984 195 8 A.E. Martell R.M. Smith Aminoacids Critical Stability Constants 1 1975 Plenum Press New York 9 E.R.B. Smith P.K. Smith J. Biol. Chem. 146 1942 187

The enthalpy of the reaction is found to be ΔH1 = −4.81 ± 0.10 kJ mol−1 (0.05 M phosphate buffer, pH 7.0, 298.15 K). Using values of the enthalpies of ionization of the buffer solution (ΔHibuffer = −3.98 ± 0.20 kJ mol−1) and N-acetylphenylalanine (ΔHiacphen = −2.46 ± 0.09 kJ mol−1), found experimentally, the enthalpy of the following hydrolysis reaction has been calculated ΔH2 = ΔH1 − ΔHibuff − ΔHiacphen = +1.63 ± 0.25 kJ mol−1, as applied to the reaction of undissociated compounds in aqueous solution. The enthalpy of reaction (2) is compared to the enthalpies of other hydrolysis reactions in compounds with ester bonds.

Subjecting high-purity nickel metal (mass fraction of impurities < 6 × 10−5) to chlorination in a calorimetric bomb with a microfurnace for sample heating made it possible to determine the standard molar change of internal energy at 298.15 K for the reaction: Ni(cub.) + Cl2(g) = NiCl2(hexagon.). The standard molar enthalpy of formation of NiCl2(hexagon.) at 298.15 K was calculated to be −(304.78 ± 0.16) kJ·mol−1. (Here and in the paper uncertainties are given for a 95 per cent confidence interval.)

Subjecting a high-purity niobium sample (total mass percentage of impurities was at most 5.7 × 10−3) to chlorination with gaseous chlorine in a calorimetric bomb made possible the measurement of the internal energy change at 298.15 K for the reaction: Nb(s) + 2.5Cl2(g) = NbCl5(s) Using the thus-found value, the enthalpy of formation of niobium pentachloride was found to be ΔHfo(NbCl5, s, 298.15 K) = −(797.34 ± 0.27) kJ·mol−1.

Die Änderung der inneren Energie bei 298.15 K für die Reaktion Nb( S ) + 2.5 C12 (g) = NbCl 5 ( s) wurde mit Nbhoher Reinheit in C12 ‐Atmosphäre kalorimetrisch gemessen.

Combustion calorimetry of tin (IV) and tin (II) oxides yielded the energies of the reactions: Sn(tetragonal)+O2(g) = SnO2(tetragonal) (1) SnO(tetragonal)+12O2(g) = SnO2(tetragonal) (2) and the enthalpies of formation of SnO2 and SnO were calculated. The results are: ΔH°f(SnO2, tetragonal, 298.15 K) = −(577.63±0.16)kJ mol− and ΔH°f(SnO, tetragonal, 298.15 K) = −(280.71±0.21)kJ mol−