Zinc enzymology is, compared to some other current areas of metallobiochemistry, a maturing field, but in addition to further developments of structure-function relationships it has also provided a number of surprising new results and ideas in the last few years. In fact, the number of studies makes it impossible to provide a comprehensive review of the recent literature on zinc enzymology here, and the authors therefore focus on those zinc enzymes for which structure-function relationships are possible on the basis of structural and biochemical data. This means that, with a few exceptions, only zinc enzymes for which NMR or crystal structures are available are included here. Another seemingly simple, yet experimentally sometimes complex issue concerns the choice of which metalloenzyme is a zinc enzyme. Since there is in principle no difference in chemical catalysis by low-affinity compared to high-affinity metal sites, some of these enzymes are also included in this article, especially if they are or have been discussed as zinc enzymes, or are active with zinc. 552 refs.

Recent Advances in Zinc Enzymology

Electrostatic Basis for Enzyme Catalysis

Although many methods for enzyme immobilization have been described in patents and publications, relatively few processes employing immobilized enzymes have been successfully commercialized. The cost of most industrial enzymes is often only a minor component in overall process economics, and in these instances, the additional costs associated with enzyme immobilization are often not justified. More commonly the benefit realized from enzyme immobilization relates to the process advantages that an immobilized catalyst offers, for example, enabling continuous production, improved stability and the absence of the biocatalyst in the product stream. The development and attributes of several established and emerging industrial applications for immobilized enzymes, including high-fructose corn syrup production, pectin hydrolysis, debittering of fruit juices, interesterification of food fats and oils, biodiesel production, and carbon dioxide capture are reviewed herein, highlighting factors that define the advantages of enzyme immobilization.

Industrial use of immobilized enzymes

The ethanol-producing bacterium Zymomonas mobilis was metabolically engineered to broaden its range of fermentable substrates to include the pentose sugar xylose. Two operons encoding xylose assimilation and pentose phosphate pathway enzymes were constructed and transformed into Z. mobilis in order to generate a strain that grew on xylose and efficiently fermented it to ethanol. Thus, anaerobic fermentation of a pentose sugar to ethanol was achieved through a combination of the pentose phosphate and Entner-Doudoroff pathways. Furthermore, this strain efficiently fermented both glucose and xylose, which is essential for economical conversion of lignocellulosic biomass to ethanol.

https://science.sciencemag.org/content/sci/267/5195/240.full.pdf

Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis

▪ Abstract This review discusses methods for the incorporation of quantum mechanical effects into enzyme kinetics simulations in which the enzyme is an explicit part of the model. We emphasize three aspects: (a) use of quantum mechanical electronic structure methods such as molecular orbital theory and density functional theory, usually in conjunction with molecular mechanics; (b) treating vibrational motions quantum mechanically, either in an instantaneous harmonic approximation, or by path integrals, or by a three-dimensional wave function coupled to classical nuclear motion; (c) incorporation of multidimensional tunneling approximations into reaction rate calculations.

/pdf/quantum-mechanical-methods-for-enzyme-kinetics-jdk1swec30.pdf

Quantum mechanical methods for enzyme kinetics

Catalytic properties of d-xylose isomerase from Streptomyces violaceoruber

D-Xylose isomerases from different bacterial strains were chemically modified with histidine and carboxylate-specific reagents. The active-site residues were identified by amino acid sequence analysis of peptides recognized by differential peptide mapping on ligand-protected and unprotected derivatized enzyme. Both types of modified residues were found to cluster in a region with consensus sequence: Phe-His-Asp-Xaa-Asp-Xaa-Xaa-Pro-Xaa-Gly, conserved in all D-xylose isomerases studied so far. These results are consistent with the recently published X-ray data of the enzyme active centre from Streptomyces rubiginosus showing hydrogen bond formation between Asp-57 and His-54 which locks the latter in one tautomeric form. A study of the pH-dependence of the kinetic parameters suggests the participation of a histidine group in the substrate-binding but not in the isomerization process. Comparison of the N-terminal amino acid sequences of several D-xylose isomerases further revealed a striking homology among the Actinomycetaceae enzymes and identifies them as a specific class of D-xylose isomerases.

Localization of the essential histidine and carboxylate group in d-xylose isomerases

The binding of two activating cations, Co2+ and Mg2+, and of one inhibitory cation, Ca2+, to D-xylose isomerase from Streptomyces violaceoruber was investigated. Equilibrium-dialysis and spectrometric studies revealed that the enzyme binds 2 mol of Co2+/mol of monomer. Difference absorption spectrometry in the u.v. and visible regions indicated that the environment of the first Co2+ ion is markedly different from that of the second Co2+ ion. The first Co2+ appears to have a six-co-ordinate. The conformational change induced by binding of Co2+ to the first site is maximum after the addition of 1 equivalent of Co2+ and yields a binding constant greater than or equal to 3.3 x 10(6) M-1. Binding of Co2+ to the second, weaker-binding, site caused a visible difference spectrum. The association constant estimated from Co2+ titrations at 585 nm agrees satisfactorily with the value of 4 x 10(4) M-1 obtained from equilibrium dialysis. Similarly, the enzyme undergoes a conformational change on binding of Mg2+ or Ca2+, the binding constants being estimated as 1 x 10(5) M-1 and 5 x 10(5) M-1 respectively. Competition between the activating Mg2+ and Co2+ and the inhibitory Ca2+ ion for both sites was further evidenced by equilibrium dialysis and by spectral displacement studies.

/pdf/metal-ion-binding-to-d-xylose-isomerase-from-streptomyces-468z2f0568.pdf

Metal ion binding to D-xylose isomerase from Streptomyces violaceoruber.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2870%2980562-2

Purification of Bacillus pumilus β-D-xylosidase by affinity chromatography

The kinetic parameters for the interconverting substrates D-xylose in equilibrium D-xylulose and D-glucose in equilibrium D-fructose were determined for several D-xylose isomerases, with Mg2+, Co2+ and Mn2+ as metal ion activators. The Km, kcat. and kcat./Km values are tabulated for the anomeric mixtures (observed parameters) as well as for the respective reactive species, i.e. the alpha-pyranose anomers of D-xylose and D-glucose and the alpha-furanose forms of D-xylulose and D-fructose (real parameters). The real Km values and catalytic efficiencies are more favourable for the ketose sugars (reverse reaction) than for the aldose sugars (forward reaction). Comparisons of the kinetic parameters further support the existence of two distinct groups of D-xylose isomerases. Inhibition constants for the cyclic substrate analogues 5-thio-alpha-D-xylopyranose and alpha-D-xylopyranosyl fluoride and for the acyclic substrate analogue xylitol and its dehydrated form 1,5-anhydroxylitol were determined and are discussed.

Hilda Kersters-Hilderson

Papers

Catalytic properties of d-xylose isomerase from Streptomyces violaceoruber

Localization of the essential histidine and carboxylate group in d-xylose isomerases

Metal ion binding to D-xylose isomerase from Streptomyces violaceoruber.

Purification of Bacillus pumilus β-D-xylosidase by affinity chromatography

Kinetic studies of Mg(2+)-, Co(2+)- and Mn(2+)-activated D-xylose isomerases.