Prosthetic groups analysis

The purified enzyme (43) was subjected to gel electrophoretic examination and metal and prosthetic group analysis. Analytical results are shown in Table III along with comparative values for other nitrate... 

Molecular weight calculations from amino acid or prosthetic group analysis is based on the simple fact that there must be at least one mole of any residue present per mole of protein. There may be more than one mole of any given residue present. Thus, this method yields a minimum molecular weight,... 

In most cases the electronic connection between an immobilized redox enzyme and the electrode requires a mediator to shuttle the electrons to the prosthetic group or some type of wiring that plays the same role. There are cases, however, especially those involving relatively small enzymes, where direct electron transfer takes place between the electrode and the prosthetic group or some electronic relay in the enzyme. Analysis of the catalysis responses then follows the principles described and illustrated in Section 4.3.2. Somewhat more complicated schemes are treated in references7, where illustrative experimental examples can also be found. 

A second example of a membrane-bound arsenate reductase was isolated from Sulfurospirillum barnesii and was determined to be a aiPiyi-heterotrimic enzyme complex (Newman et al. 1998). The enzyme has a composite molecular mass of 100kDa, and a-, P-, and y-subunits have masses of 65, 31, and 22, respectively. This enzyme couples the reduction of As(V) to As(III) by oxidation of methyl viologen, with an apparent Kra of 0.2 mM. Preliminary compositional analysis suggests that iron-sulfur and molybdenum prosthetic groups are present. Associated with the membrane of S. barnesii is a h-type cytochrome, and the arsenate reductase is proposed to be linked to the electron-transport system of the plasma membrane. 

In contrast to haem proteins there is no direct spectroscopic evidence for FeIV=0 involvement in catalysis by non-haem iron enzymes. Both the absence of a highly coloured prosthetic group and the short lifetimes of the proposed intermediates make the task of detection difficult. However, analysis of possible reaction pathways and the nature of the products formed has provided some indirect evidence for FeIV=0 formation, both in binuclear and mononuclear non-haem iron enzymes. 

After analysis of the protein, the dry weight (less ash) of the aliquot used for analysis should equal the combined weights of the amino acids recovered (calculated from the moles of amino acids found less 1 mole of water per mole of amino acid) plus any other known constituents (phosphate, carbohydrate, prosthetic groups, metal ions, etc.). If this is not the case (within experimental error), thorough examination of the protein for other unknown constituents should be made. Constituents of the protein may be expressed as moles per g (or 100000 g) of protein, or if the molecular weight of the protein is known, as moles per mole of protein. In some cases moles per 100 moles of amino acids or molar ratios (based on one amino acid) are used. 

In 1915, Harden and Norris observed that dried yeast, when mixed with lactic acid, reduced methylene blue and formed pyruvic acid 4). Thirteen years later Bernheim prepared an extract from acetone-dried baker s yeast, which had lactate dehydrogenase activity (5). Bach and co-workers demonstrated that the lactate dehydrogenase activity was associated with a 6-type cytochrome, which they named cytochrome 62 (6). In 1954, the enzyme was crystallized, enabling the preparation of pure material and the identification of flavin mononucleotide as a second prosthetic group (2). Since then, significant advances have been made in the analysis of the structure and function of the enzyme. Much of the earlier work on flavocytochrome 62 has already been summarized in previous review articles (7-10). In this article we shall describe recent developments in the study of this enzyme, ranging fi om kinetic, spectroscopic, and structural data to the impact of recombinant DNA technology. 

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