Franco Renosto

A simple method for calculating the dissociation constant of a receptor (or enzyme) · unlabeled ligand complex from radioligand displacement measurements

A general procedure is described for determining the dissociation constant of a receptor (or enzyme).unlabeled ligand complex (EI) by analyzing the I-dependent displacement of bound radioligand (A*) from EA*. The procedure (which involves measuring free A* in the presence of I) requires a knowledge of the total concentrations of receptor ([E]t), unlabeled ligand ([I]t) and radioligand ([A*]t), and the dissociation constant of the EA* complex. The unknown Kd is obtained from five simple, sequential calculations which are valid for either high or low affinity competitive unlabeled ligands and are independent of total receptor concentration or initial degree of saturation with A*. The procedure also provides the information needed to construct a distribution curve of all enzyme and ligand species (E, EA*, EI, A*, I) as [I]t is varied.

Choline sulfokinase of penicillium chrysogenum: Partial purification and kinetic mechanism

Abstract Choline sulfokinase (3′-phosphoadenosine 5′-phosphosulfate (PAPS):choline sulfotransferase, EC 2.8.2.6) was purified approximately 30-fold from the mycelium of Penicillium chrysogenum . The K m for PAPS is 12 μ m . The enzyme is remarkably specific for the adenosine 3′,5′ (or 2′-5′)-diphosphate moiety. 3′,5′-ADP (PAP) has a K i of 2.5 to 14 μ m (depending on the choline concentration) whereas the K i values of 3′-AMP, 5′-AMP, and 5′-ADP are at least 300-fold higher. The enzyme is also highly specific for choline ( K m = 17 μ M). Of a number of other amino alcohols tested, none were potent inhibitors and only dimethylaminoethanol served as a reasonably good substrate ( K m = 800 μm V = 35% of V with choline). Triethylaminoethanol was a significantly poorer substrate ( K m = 2800 μ M; V = 2% of V with choline). The purified enzyme is relatively stable when stored frozen in the presence of 25% sucrose. In the absence of sucrose, the maximum activity decreases and the K m for choline increases. (The K m for PAPS remains constant.) The age-inactivated enzyme can be restored to full activity (original V and K m for choline) by a 10-min preincubation with 50 m m mercaptoethanol. However, prolonged incubation (24 h) with 50 m m mercaptoethanol results in irreversible denaturation. Initial velocity studies established that the enzyme follows a sequential kinetic mechanism. Product inhibition studies suggest a rapid equilibrium random binding sequence. Choline- O -phosphate (a dead-end inhibitor) is linearly competitive with choline and a linear mixed type inhibitor with respect to PAPS. Choline analogs lacking the alcohol (or ester) group (e.g., trimethylammonium, neurine, chlorocholine) are competitive dead-end inhibitors with respect to choline but are uncompetitive with respect to PAPS. Thiocholine is a linear mixed type inhibitor with respect to PAPS, but the reciprocal plots are almost parallel. These results suggest that the analogs lacking an oxygen atom have a negligible affinity for the free enzyme and bind predominantly to the enzyme-PAPS complex.

Nitrate reductase from Penicillium chrysogenum: The reduced flavin-adenine dinucleotide-dependent reaction☆

Abstract Nitrate reductase from Penicillium chrysogenum catalyzes the FADH 2 -dependent reduction of nitrate in the absence of excess dithionite. At pH 7.2 (0.1 m potassium phosphate buffer) the FADH 2 /nitrate reciprocal plot patterns were parallel. Nitrite was competitive with nitrate and an uncompetitive inhibitor with respect to FADH 2 . FAD was competitive with FADH 2 and uncompetitive with nitrate. FMNH 2 and riboflavin H 2 also served as reducing agents and yielded the same kinetic patterns. The results are consistent with a nonclassical, two-site model in which nitrate and nitrite bind to a site different from the site at which FADH 2 and FAD bind. The following results suggest that the site at which FADH 2 binds to reduce the enzyme (and at which FAD binds in competition with FADH 2 to inhibit the reaction) is not the same high-affinity site to which FAD binds as a cosubstrate in the NADPH + FAD-dependent reaction: (a) The K m for FAD in the NADPH + FAD-dependent reaction at pH 7.2 is only 0.17 μ m , while the K i , for FAD in the FADH 2 -dependent reaction is 65 μ m . (b) FAD is the preferred flavin in the NADPH + oxidized flavin-dependent reaction. (The K m values for FMN and riboflavin are 15 and 41 μ m , respectively.) But all three oxidized flavins have about the same K i in the reduced flavin-dependent reaction (65, 80, and 165 μ m , respectively). Furthermore, (c) the K m values for FADH 2 , FMNH 2 , and riboflavinH 2 in the reduced flavin-dependent reaction are all about the same (ca. 900 μM), (d) V values (specific activities in units per milligram of protein) of the NADPH + oxidized flavin-dependent reaction in 0.1 m phosphate, pH 7.2, are in the order FAD (225) > FMN (150) > riboflavin (60), but the values in the reduced flavin-dependent reaction are in the order FADH 2 (100) 2 (150) 2 (500). Dithionite is known to reduce the cytochrome b component of nitrate reductase. But in the absence of a flavin, the specific activity of the dithionite-dependent reaction is only about 7—extremely low compared to the range of 60–500 for the nucleotide-dependent reactions. This result, together with the intersecting FAD/nitrate reciprocal plot patterns, suggests two possible models for the NADPH + FAD-dependent reactions: (a) FAD binds to two different high-affinity sites. At the “upstream” site, FAD mediates the transfer of electrons from NADPH to the cytochrome b prosthetic group. At the “downstream” site FAD plays either an additional electron transfer role or an activator role promoting the flow of electrons to nitrate. (b) There is only one high-affinity FAD site. FAD must occupy this site (even after the cytochrome is reduced) in order for electrons to flow to nitrate.

Nitrate reductase from Penicilliumchrysogenum: Kinetic mechanism at sub-optimum pH

Abstract The steady-state kinetics of the NADPH + FAD-dependent reduction of nitrate by nitrate reductase from Penicillium chrysogenum was studied at pH 6.18. At this sub-optimum pH, Vmax was about 83 units × mg protein−1 compared with 225 units × mg protein−1 at pH 7.20. All initial velocity reciprocal plot patterns at pH 6.18 as well as the NADP+/nitrate product inhibition pattern were intersecting. In contrast, the NADP(H)/nitrate plots at pH 7.20 were parallel ( Renosto, F. et al. J. Biol. Chem. 256 , 8616, 1981 ). A major effect of lowering the assay pH was to change the Km for FAD from 0.17 μM at pH 7.20 to 4 μM at pH 6.18. The results suggest that nitrate reductase has a steady-state random kinetic mechanism in which kcat in the forward direction at pH 7.20 (ca. 375 sec−1) is greater that koff for the dissociation of one or more substrates. Several observations suggest that koff for FAD is extremely small at pH 7.20.