Substrate specificities in class A beta-lactamases: preference for penams vs. cephems. The role of residue 237

Site saturation mutagenesis has been carried out at Ala-237 in RTEM-1 beta-lactamase to assess the role of this site in modulating differences in specificity of beta-lactamases for penams vs. cephems as substrates. (An Ala-237 Thr mutation had previously been shown to increase activity on cephems by about 30-80%.) Screening of all 19 possible mutants on penams and cephems revealed the even more active Ala-237 Asn mutant. Detailed kinetic analysis shows that this mutant has about four times the activity toward cephalothin and cephalosporin C as the wild-type enzyme. Both mutations reduce the activity toward penams to about 10% that of RTEM-1 beta-lactamase and lower by about 5 degrees C the temperature at which the enzyme denatures. Functional properties of the other mutants have also been surveyed. The most interesting aspect of these results is that two quite disparate amino acids, threonine and asparagine, when introduced for Ala-237, cause such similar changes in enzyme specificity while more similar residues do not alter the catalytic properties of the enzyme to such a significant degree.     

Conformational transmission in ATP synthase during catalysis: Search for large structural changes

Escherichia coli ATP synthase has eight subunits and functions through transmission of conformational changes between subunits. Defective mutation at Gly-149 was suppressed by the second mutations at the outer surface of the subunit, indicating that the defect by the first mutation was suppressed by the second mutation through long range conformation transmission. Extensive mutant/pseudorevertant studies revealed that / and / subunits interactions are important for the energy coupling between catalysis and H⁺ translocation. In addition, long range interaction between amino and carboxyl terminal regions of the subunit has a critical role(s) for energy coupling. These results suggest that the dynamic conformation change and its transmission are essential for ATP synthase.     

超声波和相转移催化下的Reimer—Tiemann反应

本文研究了超声波和相转移催化剂在Ｒｅｉｍｅｒ—Ｔｉｅｍａｎｎ反应中的应用,提出了超声波和相转移催化下Ｒｅｉｍｅｒ—Ｔｉｅｍａｎｎ反应的机理。研究结果表明：在超声波和相转移催化剂的共同作用下,二氯卡宾形成十分迅速,羟基苯甲醛产率显著提高,反应时间成倍缩短。     

Pressure affects enzyme function in organic media

Pressure affects enzyme function in nonaqueous media. Activation volumes have been determined and provide evidence that the primary effect of pressure is to enhance the stripping of water off an enzyme in polar organic solvents and leads to decreased enzymatic activity. Activation volumes of subtilisin Carlsberg in organic solvents, particularly with the enzyme hydrated, have a larger magnitude than activation volumes determined in aqueous solutions. This study provides further evidence that enzymatic activity in polar organic solvents is dominated by the interaction of enzyme-bound water with the solvent. From a practical standpoint, however, the results of this study suggest that enzymatic catalysis in organic solvents may be controlled by the combined effects of pressure and enzyme hydration. (c) 1993 John Wiley & Sons, Inc.     

Physicochemical properties of alkaline serine proteases in alcohol

The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1g/ml and that of alcalase was 48.1g/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters,l-Ala-l-Ala-(d-orl-)Pro-l-Phe-OMe andl-Ala-l-Ala-(d-orl-)Ala-l-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.     

Novel findings on the copper catalysed oxidation of cysteine

Summary The oxidation of cysteine (RSH) has been studied by using O₂, ferricytochrome c (Cyt c) and nitro blue tetrazolium (NBT) as electron acceptors. The addition of 200M Cu^II to a solution of 2mM cysteine, pH 7.4, produces an absorbance with a peak at 260 nm and a shoulder at 300 nm. Generation of a cuprous bis-cysteine complex (RS-Cu^I-SR) is responsible for this absorbance. In the absence of O₂ the absorbance is stable for long time while in the presence of air it vanishes slowly only when the cysteine excess is consumed. The neocuproine assay and the EPR analysis show that the metal remains reduced in the course of the oxidation of cysteine returning to the oxidised form at the end of reaction when all RSH has been oxidised to RSSR. Addition of Cu^II enhances the reduction rate of Cyt c and of NBT by cysteine also under anaerobiosis indicating the occurrence of a direct reduction of the acceptor by the complex. It is concluded that the cuprous bis-cysteine complex (RS-Cu^I-SR) is the catalytic species involved in the oxidation of cysteine. The novel finding of the stability of the complex together with the metal remaining in the reduced form during the oxidation suggest sulfur as the electron donor in the place of the metal ion.Abbreviations RSH cysteine - RS^– cysteine in the thiolate form - RS^· thiyl radical of cysteine - RSSR cystine - Cyt c cytochrome c - SOD superoxide dismutase - NBT nitro blue tetrazolium - NBF nitro blue formazan - DTNB 5,5-dithiobis-2-nitrobenzoic acid - DTPA diethylenetriaminepentaacetic acid Dedicated to prof. A. Ballio ob the occasion of his 75th birthday.     

Synthesis of some racemicγ-fluoro-α-amino acids

Summary Versatile three-step procedures for syntheses of seven racemi-fluoro-a-amino acids are described. Alkylation oftert-butyl N-(diphenylmethylene) glycinate with 1-bromo-2-fluoroalkanes gave N-protected aminoacid esters both in anhydrous medium using lithium-diisopropylamide as base at low temperature or in a two phase system of 50% aqueous sodium hydroxide and methylene chloride with triethylbenzylammonium chloride as the phase transfer catalyst at room temperature. Subsequent two-step deprotection with citric acid and hydrochloric acid gave the title compounds in 13–33% overall yields.Dedicated to Professor Dr.mult., Dr.h.c. Alois Haas on the occasion of his 65th birthday     

On the RNA World: Evidence in Favor of an Early Ribonucleopeptide World

A highly complex RNA world, as is sometimes presented in view of the widespread and diversified use of RNA enzymes, would have encountered many difficulties in passing to a world with catalysis mediated by proteins. These difficulties can be overcome by postulating a very early relationship between the nucleotide and the amino acid components. In particular, after asserting that some characteristics expressed by (nucleotide) coenzymes in catalysis are easier to understand if a close and early relationship between these coenzymes and amino acids is hypothesized, a model is presented for the origin of the enzyme–coenzyme complex. This model is essentially based on an intermediate formed by a tRNA-like molecule covalently linked to a polypeptide. The model attributes the majority of the catalytic role in the ribonucleoprotein world to the latter complex and thus it takes into account the birth of the key intermediate in the origin of protein synthesis—namely, peptidyl-tRNA, which would have otherwise been extremely difficult to select. The predictions of the model are discussed along with its robustness, using the data derived from the study of intermediary metabolism and those from molecular biology. Finally, the appearance of the genetic code in the late phase of the ribonucleopeptide world is discussed. Received: 13 January 1997 / Accepted: 25 July 1997     

Extractive bioconversions in aqueous two-phase systems: enzymatic hydrolysis of casein proteins

Partition coefficients in poly(ethylene glycol)/dextran aqueous two-phase systems are reported for mixed-casein and its components, alpha, beta and kappa casein. Rates of casein proteolysis by alpha-chymotrypsin and by trypsin are reported in single-phase and aqueous two-phase reactor systems. The advantages resulting from selective partitioning of substrates, enzymes, and products are examined in terms of relative volumetric reaction rates.