Cuatrecasas, P.

J. Biol. Chem. 1967, n/a

A spectrophotometric assay is described for staphylococcal nuclease, based on the increase in absorbance at 260 mp which accompanies deoxyribonucleic acid and RNA hy- drolysis. Initial velocities are proportional to enzyme con- centration over a 70-fold range. The enzyme has greater aflinity for DNA than for RNA, and activity is greater with heat-denatured DNA than with native DNA. No inhibitory products accumulate during the reaction. The enzyme is stable at pH values as low as 0.1, and in a concentration of 0.15 mg per ml there is no loss of activity after boiling (20 min). Dilute solutions are protected from heat inactivation by a mixture of albumin and Ca++ as well as by denatured DNA. The optimum pH for RNase and DNase activities is be- tween 9 and 10, depending on the Ca++ concentration. At higher pH values, less Ca+f is required. The inhibitory effect of high Ca+f concentrations is more pronounced at higher pH values. Considerable DNase but no RNase activity results if Ca++ is replaced by Sr+f, while Fe++ and C&f cause minimal activation. A number of heavy metal cations inhibit DNase and RNase activities competitively with Ca++; Hg++, Zn++, and Cd++ are the most potent of these. Activities resulting from combinations of DNA and RNA with Ca+f or Sr+f suggest that these substrates are hy- drolyzed by the same or closely related regions on the en- zyme. Enzyme activity toward DNA and RNA is strongly in- hibited by 5’-phosphoryl (not by 2’- or 3’-phosphoryl) deriva- tives of deoxyadenylic, adenylic, and deozythymidylic acids, and deozythymidine 3’,5’-diphosphate is the most po- tent inhibitor. High activity is obtained with polyadenylic acid compared to polyuridylic acid, polycytidylic acid, and RNA. These tidings are consistent with the known action of the enzyme (cleavage of the 5’-phosphoryl ester bond), and suggest that the differential activity toward DNA and RNA results at least in part from differences in the afhnity toward the constituent bases of these nucleic acids.

Arold, S.T.; Eppinger, J.; Groll, M.

ACS Catal. 2019, 9, 11371-11380, 10.1021/acscatal.9b02896

Artificial metalloenzymes (ArMs) have high potential in biotechnological applications as they combine the versatility of transition-metal catalysis with the substrate selectivity of enzymes. An ideal host protein should allow high-yield recombinant expression, display thermal and solvent stability to withstand harsh reaction conditions, lack nonspecific metal-binding residues, and contain a suitable cavity to accommodate the artificial metal site. Moreover, to allow its rational functionalization, the host should provide an intrinsic reporter for metal binding and structural changes, which should be readily amendable to high-resolution structural characterization. Herein, we present the design, characterization, and de novo functionalization of a fluorescent ArM scaffold, named mTFP*, that achieves these characteristics. Fluorescence measurements allowed direct assessment of the scaffold’s structural integrity. Protein X-ray structures and transition metal Förster resonance energy transfer (tmFRET) studies validated the engineered metal coordination sites and provided insights into metal binding dynamics at the atomic level. The implemented active metal centers resulted in ArMs with efficient Diels–Alderase and Friedel–Crafts alkylase activities.