A cross-linked ribonuclease A derivative, Lys7-dinitrophenylene-Lys41-ribonuclease A, has been prepared and characterized for ultimate use in protein-folding experiments. Immunochemical assays and spectroscopic measurements demonstrated that the introduction of this artificial cross-link does not perturb the native conformation of ribonuclease A. The cross-linked protein exhibited a reversible thermal transition with Tm = 53 degrees C at pH 2.0, which is 25 degrees C higher than that of unmodified ribonuclease A under the same conditions. The denatured form of the cross-linked ribonuclease A has a conformational chemical potential that is 4.9 kcal/mol higher than that of the denatured form of unmodified ribonuclease A at 40 degrees C and pH 2.0, assuming that the cross-linked and the unmodified proteins have the same conformational chemical potential in the native conformation. This is in good agreement with a theoretical value of 5.2 kcal/mol, calculated from the reduction of chain entropy of the denatured form upon introduction of the extrinsic cross-link. Thus, it is concluded that the extrinsic cross-link between Lys7 and Lys41, formed by the dinitrophenylene group, does not affect the native conformation of ribonuclease A but destabilizes the denatured conformation, probably by decreasing its chain entropy.

Porcine kidney leucine aminopeptidase has been obtained from commercial sources as in inhomogeneous preparation with variable metal content and purified by affinity chromatography over L-leucylglycyl-AH-Sepharose. Treatment with Zn2+ followed by gel filtration restores the Zn2+ content of the native enzyme, which is 6 mol of Zn2+ per hexamer, each of which is located in a single catalytic binding site per subunit. The activity of the native enzyme is modulated by incubation with divalent metal ions; it is activated by Mn2+ and Mg2+ and inhibited by Ni2+, Cu2+, Zn2+, Hg2+ and Cd2+. These metals modulate the activity by binding to a separate site on each subunit, referred to as the regulatory site. Binding of these metals at the regulatory site alters the activity of the enzyme by changing kcat, leaving KM unaltered. The number and nature of the metal binding sites of porcine kidney leucine aminopeptidase are very similar to those of the enzyme from bovine lens.

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Stopped-flow fluorescence experiments have been carried out at 23 degrees C to study the hydrolysis of Leu-Gly-NHNH-Dns [Dns = 5-(dimethylamino)naphthalene-1-sulfonyl] and Leu-Gly-NH(CH2)2NH-Dns by porcine kidney cytosol leucine aminopeptidase (LAP). Experiments have been performed with LAP species containing Mg(II), Mn(II), Ni(II), Cu(II), Zn(II), and no metal ion at the regulatory metal binding site. The fluorescence changes observed on hydrolysis of these dansyl substrates by LAP arise from changes in the concentration of substrate. Several kinetic relationships have been developed that allow the steady-state kinetic parameters for these reactions to be determined from the stopped-flow fluorescence traces. When any of the five metal ions are bound at the regulatory site, kcat and KM are both raised to approximately the same extent with the result that the maximum increase observed for kcat/KM is only approximately twofold. The effects of these metal ions on kcat, KM, and kcat/KM observed for these substrates differ markedly from those for less physiologically relevant substrates, such as Leu-p-nitroanilide, that do not have amino acids on both sides of the scissile bond. This suggests that earlier conclusions regarding the effect of the regulatory metal ion on the activity of LAP may have been misleading and casts doubt as to whether the term "regulatory site" has validity in the context of LAP-catalyzed reactions under physiological conditions.

Stopped-flow fluorescence experiments have been carried out to study the steady-state kinetics of hydrolysis of Leu-Gly-NHNH-Dns [Dns = 5-(dimethylamino)naphthalene-1-sulfonyl] by porcine kidney cytosol leucine aminopeptidase (LAP) in 50% v/v methanol/buffer solution at ambient temperature and the pre-steady-state kinetics of this reaction in the -35 to 0 degrees C temperature range. Experiments have been carried out on LAP species containing Mg(II), Mn(II), Cu(II), Ni(II), Zn(II), and no metal ion at the regulatory metal binding site. At ambient temperatures, the stopped-flow fluorescence changes observed on hydrolysis of the substrate have been used to measure the steady-state kinetic parameters kcat and KM. The results show that 50% v/v methanol lowers the values of kcat from 2- to 12-fold compared to the reactions in the absence of methanol for all of the metallo-LAP, but that the values of KM are essentially unaffected. The pre-steady-state reactions carried out under nonturnover conditions at -35 degrees C reveal a new relaxation for LAP species with Ni(II), Cu(II), and Zn(II) in the regulatory site. The value of kobsd for this relaxation reaches a plateau at high substrate concentrations, and the magnitude of its fluorescence change at a fixed concentration of substrate is proportional to the enzyme concentration. Thus, this relaxation corresponds to the production and decay of a new enzyme-substrate intermediate not observed at higher temperatures whose fluorescence differs from that of the succeeding intermediate that is normally seen above -26 degrees C.