Non-enzymatic Conditions Research Articles

The Chemical Evolution of a Nitrogenase Model, XIX Simulation of the Enzymatic Reduction of Cyclopropene

Abstract The nonenzymatic reduction of cyclopropene in molybdothiol-and related model systems of nitrogenase yields cyclopropane and propylene in a pH-dependent fashion: In alkaline media, cyclopropane is formed exclusively. In acidic solutions, cyclopropane and propylene are produced. Cyclopropene is thus identified as a pH sensitive chemical probe of nitrogenase. Since substantial amounts of propylene are formed in the enzymatic reduction of cyclopropene, it follows that the active site in functioning nitrogenase is in a locally acidic environment. The protons required to sustain the acidic pH are presumably generated by ATP hydrolysis; addition of substrate amounts of ATP to nonenzymatic cyclopropene reducing systems also stimulates propylene-and cyclopropane production. The stereochemical course of cyclopropene reduction to cyclopropane is exclusively cis, both under enzymatic and nonenzymatic conditions. The formation of propylene from cyclopropene is not stereoselective and mechanistically consistent with acid catalyzed ring opening prior to reduction.

Nonenzymatic simulation of nitrogenase reactions and the mechanism of biological nitrogen fixation.

AbstractThe development of biologically relevant model systems of nitrogenase permitted the simulation of virtually all known reactions of the nitrogen reducing enzymes under nonenzymatic conditions. On the basis of these experiments, a mechanism of biological nitrogen fixation is formulated which is in accord with the available enzymological evidence. The key reactions of the substrates of nitrogenase occur at a molybdenum active site. The non‐heme iron, which is bound to sulfur and protein‐S⊝ groups, mediates the transport of electrons to the molybdenum active site but does not participate directly in the reduction of the substrates. ATP is required for the acceleration of the reduction and activation of the molybdenum site and is hydrolyzed to ADP and inorganic phosphate. Diimine and hydrazine were detected as intermediates in the reduction of molecular nitrogen under nonenzymatic conditions.

Binding of N-acetylphenylalanyl tRNA to ribosomes—Comparison with the binding of phenylalanyl tRNA

The binding of N-acetylphenylalanyl tRNA to ribosomes (70-S) and their subunits (30-S and 50-S) in the presence of poly U was compared with that of phenylalanyl tRNA under the nonenzymatic conditions. The binding capacity of ribosomes for N-acetyl phenylalanyl tRNA was approximately one-half of that of ribosomes for phenylalanyl tRNA at low Mg 2+ concentrations. In addition, when phenylalanyl tRNA was added to the ribosome-poly U complex carrying N-acetylphenylalanyl tRNA, the amount of total bound tRNA became almost equal to the amount of bound tRNA observed using phenylalanyl tRNA alone. On the other hand, 30-S subunits bound N-acetylphenylalanyl tRNA to the same degree as phenylalanyl tRNA. Furthermore, when 50-S subunits were added under conditions where most of active 30-S subunits had N-acetylphenylalanyl tRNA, the additional binding of phenylalanyl tRNA to this complex took place, but 50-S subunits did not stimulate the additional binding of N-acetylphenylalanyl tRNA.

Mechanisms in protein synthesis IV. Further evidence for two different ribosomal sites, one binding formylmethionyl-tRNA, the other methionyl- and other aminoacyl-tRNA's

The binding of N-acetylphenylalanyl tRNA to ribosomes (70-S) and their subunits (30-S and 50-S) in the presence of poly U was compared with that of phenylalanyl tRNA under the nonenzymatic conditions. The binding capacity of ribosomes for N-acetyl phenylalanyl tRNA was approximately one-half of that of ribosomes for phenylalanyl tRNA at low Mg2+ concentrations. In addition, when phenylalanyl tRNA was added to the ribosome-poly U complex carrying N-acetylphenylalanyl tRNA, the amount of total bound tRNA became almost equal to the amount of bound tRNA observed using phenylalanyl tRNA alone. On the other hand, 30-S subunits bound N-acetylphenylalanyl tRNA to the same degree as phenylalanyl tRNA. Furthermore, when 50-S subunits were added under conditions where most of active 30-S subunits had N-acetylphenylalanyl tRNA, the additional binding of phenylalanyl tRNA to this complex took place, but 50-S subunits did not stimulate the additional binding of N-acetylphenylalanyl tRNA.

A theory of the mechanism of enzyme action.

A LTHOUGH there exists an extensive literature concerned with the nature of enzyme action and the chemistry and structure of enzymes, little progress has been made toward understanding the mechanism of enzyme action in explicit terms of the molecular structure of proteins, prosthetic groups, and their combinations. Woolf (1931), Szent-Gyorgyi (1941, 1947), and LuValle and Goddard (1948) are among those who have dealt with the problem and have proposed mechanisms, but while they have treated in detail certain of the fundamental aspects of enzyme action, they have given only cursory consideration to the structure and role of the protein components of enzymes. There can be little doubt that the protein portion of the enzyme molecule plays an essential role in its reactions. Indeed, many enzymes consist, so far as is known, entirely of protein. Enough is known at the present time of the structure of enzymes, of the nature of enzymatic reactions, and of the kinds of substrates and products involved in enzymatic processes to justify the attempt to evolve a general hypothesis of the mechanism of these reactions. Except for the participation of inorganic ions, it is evident that enzymes are primarily organic compounds and that, in the majority of cases, they act upon organic substances, the reactions involving for the most part organic oxidations, reductions, hydrolyses, and condensations. In the present treatment the approach will be made from the following point of view: that reactions involving organic substances can be examined from the standpoint of present-day concepts of the nature and mechanisms of organic reactions. It will be shown in this paper that it is possible to consider all enzyme reactions to involve the same fundamental property of the protein molecule, and that when prosthetic groups are essential participants in the process, as in oxidation-reduction reactions, the role of the prosthetic group is auxiliary and not an independent one. In short, it is suggested that enzyme reactions of all kinds have as a common denominator a unique and characteristic property of the protein molecule, which lies in its possession of a long hydrogenbridged system of peptide linkages. The question of the mechanism of enzyme action is essentially one of the means by which the enzyme brings about a sufficient lowering of the activation energy of a normally slow chemical reaction to cause its rate to be enormously increased over what it would be under non-enzymatic conditions and at comparable conditions of pH and temperature (see Kalckar, 1946).