The kinetics and specificity of the reaction of 2'(3')-O-bromoacetyluridine with bovine pancreatic ribonuclease A.

2'(3')-O-Bromoacetyluridine reacts rapidly and selectively with bovine pancreatic ribonuclease A at pH 5.5 and 25 degrees. Under conditions of high molar ratios of nucleoside derivative to enzyme, the only derivative is N-3-carboxymethylhistidine-12 ribonuclease A. The reaction occurs almost exclusively with the histidine-12 residue at the active site inactivation of the enzyme is accompanied by the stoichiometric disappearance of unmodified ribonuclease A and appearance of the product, N-3-carboxymethylhistidine-12 ribonuclease A. Kinetic studies indicate a mechanism involving saturation of the enzyme by the nucleoside derivative. The inhibitor constant, Kb, is 0.087 M and k3 is 35.1 times 10(-4) sec minus 1. The reaction of 2'(3')-O-bromoacetyluridine with the enzyme occurs at a rate approximately 3100 times greater than that corresponding to the reaction with L-histidine. The alkylation reaction is inhibited competitively by uridine with a Ki of 0.013 M. 2'(3')-O-Bromoacetyluridine inactivates ribonuclease A 4.5 times faster than bromoacetic acid and the specificity for alkylation of active-site histidine residues is different. 2'(3')-O-Bromoacetyluridine reacts 1000 times more rapidly with ribonuclease A than iodoacetamide. The contribution of nucleoside binding to the overall rate of alkylation is discussed.     

Crystal packing in six crystal forms of pancreatic ribonuclease.

We compare the molecular packing of bovine pancreatic ribonuclease A (RNase A) in six crystal forms, two grown with alcohol, three with high salt and one with polyethylene glycol as a precipitant. The six packings differ in the number of molecules in contact and in the extent of the contacts, which bury 1570 A2 to 2790 A2 of the RNase surface. Regions of the protein surface involved in the six packings cover almost the whole RNase molecule. The abundance of polar interactions, about one per 200 A2, is the same in all types of precipitants. All molecule-to-molecule contacts are different in the six crystal forms, except for the one that forms a RNase dimer. The dimer has a large interface covering 1800 A2 and eight to ten polar interactions. Its presence in the three salt-grown crystal forms suggests that it is an intermediate in salt induced crystallization. In contrast, the two alcohol-grown forms contain only small interfaces, implying a different mechanism of nucleation.     

The kinetics of the recombination reaction between apomyoglobin and alkaline haematin.

Apomyoglobin was prepared by an extremely mild modification of the acid/butanone technique, and the kinetics of the recombination reaction between this preparation and alkaline haematin were studied. The recombination has been shown to be precisely second-order and mono-phasic. Rate constants obtained from the study are in good agreement with values obtained previously by an indirect technique not involving separation of haem and apoprotein.     

The kinetics of the reaction of nitrophenyl phosphates with alkaline phosphatase from Escherichia coli

1. The steady-state rate of hydrolysis of 2,4-dinitrophenyl phosphate catalysed by Escherichia coli phosphatase is identical with that of 4-nitrophenyl phosphate over the pH range 5.5-8.5. 2. The increase in the rate of the enzyme-catalysed decomposition of nitrophenyl phosphates in the presence of tris at pH8.1 and 5.9 is consistent with the hypothesis that tris increases the rate of decomposition of a phosphoryl-enzyme intermediate. At pH8.1 the rate of decomposition of the phosphoryl-enzyme is approximately twice as fast as the rate of its formation, whereas at pH5.9 the rate of formation of the phosphoryl-enzyme is considerably faster than its decomposition. 3. Pre-steady-state measurements of the initial transient of the liberation of 2,4-dinitrophenol during the reaction of the enzyme with 2,4-dinitrophenyl phosphate confirmed the above pH-dependence of the ratio of the rates of phosphorylation and dephosphorylation of the enzyme. At optimum pH (above pH8), when the phosphorylation of the enzyme by the substrate is rate-determining, this step must be controlled by a rearrangement of the enzyme or enzyme-substrate complex.     

The molecular evolution of pancreatic ribonuclease

Summary The primary structures of pancreatic ribonucleases from 26 species (18 artiodactyls, horse, whale, 5 rodents and turtle) are known. Several species contain identical ribonucleases (cow/bison; sheep/goat), other species show polymorphism (arabian camel) or the presence of two structural gene loci (guinea pig pancreas contains two ribonucleases that differ at 31 positions). 26 different sequences (including the ribonuclease from bovine seminal plasma which is paralogous to the pancreatic ribonucleases) were used to construct a most parsimonious tree. A second tree that most closely approximates current biological opinion requires 402 whereas the most parsimonious tree requires 389 nucleotide substitutions. The artiodactyl part of the most parsimonious tree conforms quite well with the biological one of this order, except for the position of the giraffe which is placed with the pronghorn. Other parts of the most parsimonious tree agree less with the biological tree, probably as a result of the occurrence of many parallel and back substitutions. Bovine seminal ribonuclease was found to be the result of a gene duplication which occurred before the divergence of the true ruminants, but after the divergence of this group from the cameloids.The evolutionary rate of ribonuclease was found to be 390, 3.0 and 11 nucleotide substitutions per 10⁹ yrs per ribonuclease gene, codon and covarion respectively. However, there is much variation in evolutionary rate in different taxa. Values ranging from about 100 (in the bovidae) to about 700 (in the rodents) nucleotide substitutions per 10⁹ yrs per gene were found.A method for counting parallel and back mutations is presented. The 389 nucleotide substitutions in the most parsimonious tree occur at 88 codon positions; 154 of them are the result of parallel and back mutations. Parallel evolution to a similar structure, including the presence of 2 sites with carbohydrate, was demonstrated in an extensive region at the surface of pig and guinea pig ribonuclease B. The presence of carbohydrate probably is important in a number of species. A correlation between the presence of heavily glycosidated ribonucleases and coecal digestion was observed. Hypothetical sequences of ancestral ungulate ribonucleases contain many recognition sites for carbohydrate attachment; this suggests that herbivores with coecal digestion might have preceded the true ruminants in mammalian evolution.     

Kinetic studies on the cleavage of oligouridylic acids and poly U by bovine pancreatic ribonuclease A

Kinetic parameters, Km and Vmax for the transesterification of oligouridylic acid, (Up)nU greater than p (n=0-4), by RNase A were measured spectrophotometrically at pH 7.0 and 25 degrees C. The kinetic parameters, pKm and log Vmax increased with increase in the chain length (n), and seemed to be almost constant with substrates having n greater than or equal to 2. The contribution of each subsite to the binding was estimated according to Hiromi's theory. The subsite affinities for (B1, R1, P1)+(B2, R2, P2) and (B3, R3, P3) are 8.03 kcal and 0.72 kcal/mol, respectively, and those for (B4, R4, P4) and (B5, R5, P5) are less than 0.5 kcal/mol. Therefore, we postulate that the size of the RNase A active site is about 3 nucleotides in length. Transesterification of poly U by RNase A was followed spectrophotometrically. The reaction is markedly influenced by ionic strength. At lower ionic strength, the v0-S curve of poly U cleavage was sigmoidal and cooperative, and it became less cooperative at higher ionic strength. Since the estimated Vmax value for poly U cleavage at ionic strength of 0.1 was more than 20 times larger than that of oligouridylic acids cleavage, we propose a non-specific interaction of poly U anion with cationic groups on the surface of the enzyme, modulating the conformation of active site, and thus increasing the activity at low ionic strength. The interaction decreases at higher ionic strength due to the interaction of counter anions with the non-specific sites.     

The reaction of ribonuclease inhibitor with modified ribonucleases

1. The effect of chemical modification of ribonuclease on its reaction with ribonuclease inhibitor has been studied. 2. Removal of free amino groups from the enzyme with nitrous acid or by acetylation did not affect the reaction. Some changes altered the stoicheiometry of the reaction and ribonuclease S was found to be inhibited linearly by increasing amounts of ribonuclease inhibitor, in contrast with ribonuclease A, which is inhibited in a non-linear way. One derivative of ribonuclease containing dimethylaminonaphthalenesulphonyl groups actually reacted with ribonuclease inhibitor to a greater extent (and linearly) than did the unaltered enzyme. 3. The positively charged histidine at the active site and the active enzyme did not appear to be necessary for the reaction since 1-carboxymethylhistidine-119-ribonuclease reacted with ribonuclease inhibitor to almost the same extent as the native enzyme. In general, any significant change in the conformation of ribonuclease was accompanied by a loss in its ability to combine with inhibitor. The presence of 8m-urea also prevented reaction of ribonuclease with inhibitor. 4. Some characteristics of the reaction of ribonuclease inhibitor, ribonuclease and deaminated ribonuclease with RNA and deaminated RNA were investigated.