Using Steady-State Kinetics to Quantitate Substrate Selectivity and Specificity: A Case Study with Two Human Transaminases

We examined the ability of two human cytosolic transaminases, aspartate aminotransferase (GOT1) and alanine aminotransferase (GPT), to transform their preferred substrates whilst discriminating against similar metabolites. This offers an opportunity to survey our current understanding of enzyme selectivity and specificity in a biological context. Substrate selectivity can be quantitated based on the ratio of the k_cat/K_M values for two alternative substrates (the ‘discrimination index’). After assessing the advantages, implications and limits of this index, we analyzed the reactions of GOT1 and GPT with alternative substrates that are metabolically available and show limited structural differences with respect to the preferred substrates. The transaminases’ observed selectivities were remarkably high. In particular, GOT1 reacted ~10⁶-fold less efficiently when the side-chain carboxylate of the ’physiological’ substrates (aspartate and glutamate) was replaced by an amido group (asparagine and glutamine). This represents a current empirical limit of discrimination associated with this chemical difference. The structural basis of GOT1 selectivity was addressed through substrate docking simulations, which highlighted the importance of electrostatic interactions and proper substrate positioning in the active site. We briefly discuss the biological implications of these results and the possibility of using k_cat/K_M values to derive a global measure of enzyme specificity.     

Comparative Study of Substrate- and Stereospecificity of Penicillin G Amidases from Different Sources and Hybrid Isoenzymes

Summary.  Four natural pencillin G amidase variants from different sources and two genetically constructed hybrid enzymes were produced and purified to homogeneity. The specificity constants of one enzyme (E. coli) were found to differ six orders of magnitude for hydrolytic transformations within a wide range of substrates. The substrate specificity of the homologous penicillin amidases was found to differ less than one order of magnitude for hydrolysis of the most specific and up to two orders of magnitude for the less specific substrates. The -substrate specificity in hydrolytic and transfer reactions (studied mainly with the E. coli enzyme) varied more than three orders of magnitude for the different substrates. The penicillin amidases were found to be R-specific in the S ₁-binding site and S-specific in the -binding site. The S ₁-stereoselectivity differs less than one order of magnitude for the different variants. The -stereoselectivity is more pronounced, increases with nucleophile specificity, and was found to differ up to three orders of magnitude in transfer reactions for the enzyme from E. coli. The observed variation of enatioselectivity for different penicillin amidases and one substrate can also be achieved by changes in temperature. Comparison of substrate- and stereospecificity of penicillin amidases from different sources and hybrid isoenzymes suggests that similar changes can be expected for enzyme variants derived by rational protein design or directed evolution. Received December 20, 1999. Accepted (revised) February 4, 2000     

组织工程相关生物材料表面工程的研究进展

生物材料用作人工细胞外基质(ECM ) 在组织工程中占据重要位置。本文在分析细胞2生物材料表面相互作用的基础上, 从生物材料中的水、材料表面的形态、材料表面的特异性识别及生物材料诱发愈合等方面探讨了生物材料的复杂性。生物材料对细胞的影响是一个双向、动态过程, 起着调节细胞增殖和凋亡平衡的作用。基于生物材料对细胞生长的影响, 本文提出了生物材料表面生物仿生化以提高细胞亲和力,糖链团簇、糖脂质及材料表面蛋白质修饰以提高细胞特异性识别, 材料表面的自组装修饰以改善表面形态等观点。     

Substrate specificity of the human proteasome

BACKGROUND: Regulated proteolysis by the proteasome is crucial for a broad array of cellular processes, from control of the cell cycle to production of antigens. RESULTS: The rules governing the N-terminal primary and extended substrate specificity of the human 20S proteasome in the presence or absence of 11S proteasome activators (REGalpha/beta and REGgamma) have been elaborated using activity-based proteomic library tools. CONCLUSIONS: The 11S proteasome activators are shown to be important for both increasing the activity of the 20S proteasome and for altering its cleavage pattern and substrate specificity. These data also establish that the extended substrate specificity is an important factor for proteasomal cleavage. The specificities observed have features in common with major histocompatibility complex (MHC) class I ligands and can be used to improve the prediction of MHC class I restricted cytotoxic T-cell responses.     

Composition analysis of positional isomers of phosphatidylinositol by high-performance liquid chromatography

An HPLC-based method has been developed for composition analysis of six positional isomers of phosphatidylinositol (PI), of which the phosphatidyl group was connected to different positions of the myo-inositol moiety. The method employed a combination of two types of HPLC analyses. One was direct separation of the six PI isomers into four peaks of 1(3)-PI, 2-PI, 4(6)-PI and 5-PI on a normal-phase silica gel column. The second method was for the separations of 1-PI from 3-PI and 4-PI from 6-PI, which were not separable on the normal-phase column. This method involved conversion of PI isomers into pentakis-(R)-1-phenylethylcarbamate (PEC) derivatives, which were separated on a reversed-phase column. Using the established method, positional specificity of several engineered phospholipases D in enzymatic synthesis of PI from myo-inositol and phosphatidylcholine was investigated. This was performed by analyzing the isomeric composition of PIs synthesized by the mutant enzymes. Among five mutant enzymes tested, two showed strong specificity to 1-OH, one showed moderate preference to 1-OH, one preferred 3-OH, and one showed broad specificity towards 1-, 3-, 4- and 6-OH.     

Immunoassay for the detection of lead ions in environmental water samples

A rapid, simple, and reliable competitive immunoassay was developed for measurement of lead ions Pb(II) in environmental samples. Avian antibodies were produced against Pb(II). Since lead ions are too small to elicit an immune response, the metal was coupled to protein carrier Bovine serum albumin (BSA) using a bifunctional chelator 1-(4-isothiocyanobenzyl) ethylenediamine N,N,N′,N′-tetra acetic acid (ITCBE). Poultry birds (layers) were immunised with this Pb(II)–ITCBE–BSA immunoconjugate and the avian antibodies (IgY) isolated from egg yolk recognised Pb(II)-ITCBE complexes as capture reagent and a Pb(II)–ITCBE conjugate of Alkaline phosphatase as an enzyme label. Antibody reaction was optimised for different concentrations of antigen and antibody dilutions. Cross reactivity with other metals were below 1% in competitive ELISA. The IC₅₀ value of this avian antibody was 0.19?µg?mL^?1. The detection range and the detection limit were 0.02–1000?µg?mL^?1and 0.2?µg?mL^?1, respectively.     

D-lactate dehydrogenase

This note compares the substrate specificity of D-lactate dehydrogenase (D-LDH, EC 1.1.1.28) to that of L-lactate dehydrogenase (L-LDH, EC 1.1.1.27), illustrates three procedures that use D-LDH in synthesis and two methods for recycling NADH, and provides experimental details illustrating the use of D-LDH in organic synthesis.     

Gels mimicking antibodies in their selective recognition of proteins

Summary In a previous paper we presented preliminary experiments aimed at the preparation of gel particles with the property to recognize selectively some particular protein (hemoglobin, cytochrome C, transferrin) [1]. Using the same method we show in this article that human growth hormone, ribonuclease and myoglobin from horse can also be adsorbed specifically, indicating that the method may be universal or at least applicable to a great number of proteins. A gel with specific adsorption of three model proteins was synthesized in order to demonstrate that the beds can be employed to remove (traces of) several proteins contaminating a sample (“negative purification”). The degree of selective recognition is high, to judge from the fact that myoglobin from horse, but not that from whale, was adsorbed onto a column designed to bind specifically the former protein. This selectivity is noteworthy, since these two proteins have similar amino acid sequences and 3-D structures. The method for the synthesis of the specific gels involves polymerization of appropriate monomers (for instance acrylamide and its derivatives) in the presence of the protein to be adsorbed specifically, granulation of the gel formed, packing a column with the gel particles, washing the column to remove the protein and finally application of the sample for selective adsorption of the protein. The approach resembles that used for entrapment (immobilization) of proteins for affinity chromatography and that for molecular imprinting, with the distinct difference that the monomer composition is quite different and thereby the binding mechanism. This mechanism is discussed, for instance, in terms of (1) a new classification system for chromatographic beds based on the number of bonds between the solute and the matrix and the strength of each bond and (2) “non-specific bonds” (these bonds are often harmful in conventional chromatography, but we have used them to advantage). In this classification system the selective recognition is characterized by a large number of weak bonds. Therefore, so-called functional monomers are not used for the preparation of the gels because they often are charged and, accordingly, give rise to strong electrostatic interactions, i.e. the beds behave to some extent as ion-exchangers. In most experiments we have used a polyacrylamide gel with large pores to facilitate diffusion of proteins into and out of the gel granules. When used in chromatography these soft gels (which can be used repeatedly) allow only rather low flow rates. This problem can be overcome by a new approach to prepare the granules. Potential applications of the selective beds are discussed, as well as future improvements. YW's visit to the Department was sponsored by the Swedish Institute, Stockholm, Sweden.     

Characterization of an Isozyme of β-Glucosidase from Sweet Almond

A sweet almond β-glucosidase (EC 3.2.1.21) isozyme was purified from commercial crude product. The process of purification consisted of a Protein-Pak Q anion exchange chromatography following by a Superdex 75 HR gel filtration separation. The purified enzyme is a monomeric glycoprotein with molecular weight of 58 kDa and pI=4.55 which is distinguished from reported isozymes. The enzyme has apH optimum in the range of 5.2-5.6 when p-nitrophenyl-β-D-glycopyranosides are used as substrate and is stable up to 50 °C at that pH range. The purified protein also exhibits profound β-galactosidase and σ-L-arabinosidase activity. The study of substrate specificity revealed that lacking of hydroxymethyl group at C-5 of glycosides resulted in higher affinity for substrate binding to enzyme, whereas the chemical step of hydrolysis (k_cst) was prevented significantly. The pH activity profile displayed a bell-shaped curve for all measured p-nitrophenyl-β-D-glycopyranosides with apparent pK₁ and pK₂ values of 4.4-4.7 and 6.2-6.4, respectively. This isozyme was strongly inhibited by δ-gluconolactone (K_i = 160 μM) and 4-phenylimidazole (K_i = 17.8 μM) reversibly at pH 6.2. Among the tested glycoses, the binding affinity of N-acetyl-β-D-glucosamine to the enzyme (K_l = 52 mM) was 6 times stronger than that of glucose and its epimers.     

Preparation and evaluation of OV-240-OH coated glass capillary columns for isomer specific determination of PCDDs and PCDFs

Glass capillaries were leached, dehydrated, persilylated with 1, 3-bis (3-cyanopropyl) tetramethyldisiloxane, and coated with OV-240-OH. After crosslinking and binding the phase to the glass surface the columns showed high separation efficiency, high temperature stability, and inertness comparable to persilylated apolar columns. Column performance is shown to be superior to liquid phase cyanopropyl columns such as SP 2330. The excellent separation capabilities together with the selectivity of the phase makes OV-240-OH coated columns a valuable tool for the determination of toxic isomers in complex mixtures of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). The order of elution of individual TCDD isomers was found to be similar to that described for SP 2330 or Silar 10c. The detection of PCDDs and PCDFs in a fly ash extract further illustrates the utility of OV-240-OH coated columns. The high temperature limit of these columns opens the way for the analysis of high boiling compounds such as mixed brominated/chlorinated dibenzo-p-dioxins and dibenzofurans.