Apoenzyme-cofactor interactions

Enzymes may not function well or at all unless some other species known as a cofactor is present. An enzyme alone is referred to as the apoenzyme and the combination of enzyme and cofactor is known as the holoenzyme. Among the species that function as cofactors are organic compounds that interact with the enzyme. If the organic moiety is strongly attached to the enzyme, it is called a prosthetic group, but if it is loosely bound to the enzyme, it is referred to as a coenzyme. For the purposes of this discussion, the most interesting cofactors are metal ions. Depending on the type of enzyme, the appropriate metal ion cofactor may be Mg2+, Ca2+, K+, Fe2+, or Cu2+. A sizeable number of enzymes are sometimes called metalloenzymes because they have active sites that contain a metal. 

A model of a flavin-based redox enzyme was prepared.[15] Redox enzymes are often flavoproteins containing flavin cofactors flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). They mediate one- or two-electron redox processes at potentials which vary in a range of more than 500 mV. The redox properties of the flavin part must be therefore tuned by the apoenzyme to ensure the specific function of the enzyme. Influence by hydrogen bonding, aromatic stacking, dipole interactions and steric effects have been so far observed in biological systems, but coordination to metal site has never been found before. Nevertheless, the importance of such interactions for functions and structure of other biological molecules make this a conceivable scenario. 

S. The lack of stoichiometry between cofactor and apoenzyme interaction is analogous to the variable metal content. 

Although the catalytic activity of some enzymes depends only on interactions between active site amino acids and the substrate, other enzymes require nonprotein components for their activities. Enzyme cofactors may be ions, such as Mg2+ or Zn2+, or complex organic molecules, referred to as coenzymes. The protein component of an enzyme that lacks an essential cofactor is called an apoenzyme. Intact enzymes with their bound cofactors are referred to as holoenzymes. 

S-labelled Molybdenum Cofactor. The possibility of direct incorporation of S into PPT using controlled in vitro synthesis with purified proteins was recently used to reconstitute a S-labelled cofactor into the apoenzyme. While no spectral difference could be detected on the X-band CW-EPR spectra of the IpH samples prepared with S- and S-labelled MPT, the use of primary and refocused primary ESEEM at 35 GHz allowed the detection of the interdoublet transition from a S nucleus with strong nuclear quadrupole interaction. The estimated hfi and nqi parameters (Aj o ( S) = 3 MHz, Ti,2,3 ( S) = [1, -1, 0] MHz, (e qQ/h, t]) ( S) = (25 MHz, 0.5) and Euler angles of the quadrupolar tensor in the g-frame (a, p, y) = (0°, 40°, 0°)) are in good agreement with those predicted by DFT for the two indistinguishable... 

A variation of the EMIT is the apoenzyme reconstruction immunoassay system, where the analyte is coupled not to the enzyme itself but to a cofactor or prosthetic group that is required for enzyme activity. For example, the analyte may be coupled to flavin-adenine dinucleotide (FAD), which is required for glucose oxidase activity. The antibody binds to the FAD-analyte conjugate and prevents FAD from interacting with the enzyme, whose activity is consequently reduced. The more analyte present in the sample, the more competition there is for the antibody and the more FAD-analyte conjugate remains free in solution and available to the enzyme. Again, the level of enzyme activity is related to the quantity of analyte in the sample. 

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