Allosterism, regulation and cooperativity: The case of ribonucleotide reductase of Lactobacilus leichmannii

Abstract The binding of modifiers and substrates to the ribonucleotide reductase from Lactobacillus leichmannii has been studied with the use of enzyme highly purified by a modified preparative procedure. This monomeric enzyme represents a simpler system than classical allosteric enzymes since the study of allosterism, regulation, and conformational responses in the latter is complicated by interaction between subunits. Analysis of earlier kinetic results indicates that ribonucleotide substrates bind weakly to the regulatory site (KD for GTP, 4 m m ) compared with binding to the catalytic site (KD for GTP 0.24 m m ), and in so doing produce substrate activation. The apparent rate constant for product formation when GTP is not bound to the regulatory site is 31% of that observed when GTP occupies the regulatory site. Part of this effect is due to tighter binding of coenzyme when substrate is bound to the regulatory site, a result confirmed by direct measurement of adenosylcobalamin binding. The effect of modifiers such as dGTP which accelerate the reduction of specific substrates is also in part due to increased coenzyme binding as measured both kinetically and by equilibrium dialysis and ultrafiltration methods. The modifiers also seem to have an obligatory role in the enzyme mechanism. Without their presence neither formation of the radical-pair intermediate from adenosylcobalamin nor associated reactions (coenzyme degradation, coenzyme H-exchange with water) occur under the experimental conditions observed. When modifier concentration dependence for these reactions is compared with estimates of equilibrium constants for dissociation of modifiers and substrates from complexes with the regulatory and catalytic sites it appears likely that binding at the regulatory site is responsible. Calculation of coenzyme saturation under the experimental conditions indicates, however, that insignificant amounts of coenzyme are bound to the enzyme except when the regulatory site is occupied, and this effect adequately explains the necessity for modifiers. Binding of dGTP does not alter the circular dichroism of the enzyme in the far-ultraviolet region (200 to 250 nm) but causes significant changes in the aromatic region, and therefore appears to perturb tryptophyl and tyrosyl residues though not necessarily directly. Measurements have been made of the fractional change in sedimentation coefficient due to dGTP binding to the enzymes by the use of an adjustable Raleigh mask and double sector cell in the Beckman model E ultracentrifuge. Values for Δs/ s of −1.14% at 22° and −1.16% at 37° were obtained. The former, after correction for the fractional increase due to the calculated effect of dGTP on the buoyant weight of the protein, gives a value of (Δs/ s corr = −2.84% . If this is due to increase in hydrodynamic volume of the protein alone, it corresponds to an 8.8% increase. If it were entirely due to increased departure from spherical shape it would correspond to a change in axial ratio from 2.9 to 3.2. In the presence of saturating concentrations of ATP and dihydrolipoate and the maximum permissible concentration of a coenzyme analogue Δs/ s was −0.33%, the corrected value being about −1.99% at 37°. These conformational responses are as great as determined for multimeric enzymes such as in the effect of CTP on aspartate transcarbamylase, and are consistent with relay of the regulatory signal over a considerable intersite distance.