The pH-dependence of second-order rate constants of enzyme modification may provide free-reactant pKa values.

1. Reactions of enzymes with site-specific reagents may involve intermediate adsorptive complexes formed by parallel reactions in several protonic states. Accordingly, a profile of the apparent second-order rate constant for the modification reaction (Kobs., the observed rate constant under conditions where the reagent concentration is low enough for the reaction to be first-order in reagent) against pH can, in general, reflect free-reactant-state molecular pKa values only if a quasi-equilibrium condition exists around the reactive protonic state (EHR) of the adsorptive complex. 2. Usually the condition for quasi-equilibrium is expressed in terms of the rate constants around EHR: (formula: see text) i.e. k mod. less than k-2. This often cannot be assessed directly, particularly if it is not possible to determine kmod. 3. It is shown that kmod. must be much less than k-2, however, if kobs. (the pH-independent value of kobs.) less than k+2. 4. Since probable values of k+2 greater than 10(6)M-1.S-1 and since values of kobs. for many modification reactions less than 10(6)M-1.S-1, the equilibrium assumption should be valid, and kinetic study of such reactions should provide reactant-state pKa values. 5. This may not apply to catalyses, because for them the value of kcat./Km may exceed 5 X 10(5)M-1.S-1. 6. The conditions under which the formation of an intermediate complex by parallel pathways may come to quasi-equilibrium are discussed in the Appendix.     

6 beta-Bromopenicillanic acid inactivates beta-lactamase I.

The inactivation of beta-lactamase I by preparations of 6alpha-bromopenicillanic acid showed unexpected kinetic features that indicated that the active species was the 6beta-epimer. Samples containing 6beta-bromopenicillanic acid have been synthesized and shown to inactivate the enzyme in a rapid stoicheiometric reaction.     

The pH-dependence of class B and class C beta-lactamases.

The classification by structure allots beta-lactamases to (at present) three classes, A, B and C. The pH-dependence of the kinetic parameters for class B and class C have been determined. They differ from each other and from class A beta-lactamases. The class B enzyme was beta-lactamase II from Bacillus cereus 569/H/9. The plots of kcat against pH for the hydrolysis of benzylpenicillin by Zn(II)-requiring beta-lactamase II and Co(II)-requiring beta-lactamase II were not symmetrical, but those of kcat/Km were. A similar feature was observed for the hydrolysis of both benzylpenicillin and cephalosporin C by a class C beta-lactamase from Pseudomonas aeruginosa. The results have been interpreted by a scheme in which two ionic forms of an intermediate can give product, but do so at differing rates.     

The pH-dependence of the peptidase activity of aminoacylase.

Aminoacylase is a potent peptidase around pH 8.5. The pH dependence of the Km values reveals that only dipeptides with uncharged N-terminal amino acids are substrates of the enzyme. The Km values reflect the hydrophobicity of the N-terminal amino acids. Calculated on the basis of unprotonated peptides they are pH independent. Hydrophobic, deprotonated amino acids are competitive inhibitors of the enzyme, tryptophan and norleucine being the strongest inhibitors. Inhibitor constants with glycylalanine as substrate have been determined for several amino acids. From the present results it may be deduced that the N-terminal amino acids of dipeptides are bound at a strongly hydrophobic site.     

The pH-dependence of pepsin-catalysed reactions

1. The pH-dependence of the pepsin-catalysed hydrolysis of three peptide substrates was studied by using a method for the continuous monitoring of the formation of ninhydrin-positive products. 2. Two peptide acid substrates, N-acetyl-l-phenylalanyl-l-phenylalanine and N-acetyl-l-phenylalanyl-l-phenylalanyl-glycine, show apparent pK(a) values of 1.1 and 3.5 in the plots of k(0)/K(m) versus pH. By contrast a neutral substrate, N-acetyl-l-phenylalanyl-l-phenylalanine amide, shows apparent pK(a) values of 1.0 and 4.7. 3. Together with the data of the preceding paper (Knowles, Sharp & Greenwell, 1969), these results are taken to indicate that the rate of pepsin-catalysed hydrolysis is controlled by the ionization of two groups, which on the free enzyme have apparent pK(a) values of 1.0 and 4.7. It is apparent that the anions of peptide acid substrates are not perceptibly bound to the enzyme, resulting in apparent pK(a) values of 3.5 for the dependence of k(0)/K(m) for these materials.     

beta-lactamase I from Bacillus cereus. Structure and site-directed mutagenesis.

The kinetic model of Ragan & Cottingham [(1985) Biochim. Biophys. Acta 811, 13-31] for electron transport through a mobile pool of quinone predicts that, under certain conditions, the normal linear dependence of electron flow on the degree of reduction (or oxidation) of the quinone should no longer be found. These conditions can be met by reconstituted NADH: cytochrome c reductase (Complex I-III from bovine heart) when electron flow is rate-limited by a low concentration of cytochrome c. We show that, in such a system, the dependence of activity (varied by inhibition with rotenone) on the steady-state level of quinone reduction is indeed non-linear and very closely accounted for by the theory.     

The antiquity of group I introns.

The recent discovery of self-splicing introns in cyanobacteria has given renewed interest to the question of whether introns may have been present in the ancestor of all living things. The properties of introns in genes of bacteria and bacteriophages are discussed in the context of their possible origin and biological function.     

Reversibility of the ampicillin-and nitrite-induced inactivation of beta-lactamase I.

beta-Lactamase I was isolated from Bacillus cereus 569/H. Treatment with ampicillin in the presence of sodium nitrite at pH 4 or 5 resulted in the inactivation of the enzyme presumably by modification of a carboxyl group in the active site. However, this inactivation was rapidly, reversible at neutral pH and the available evidence points to the participation of a second carboxyl group which is involved in the reactivation process.     

Kinetic characterization of the acyl-enzyme mechanism for beta-lactamase I.

beta-Lactamase I catalyses the hydrolysis of penicillins by an acyl-enzyme mechanism. A procedure was developed for determining the rate constants for the acylation and deacylation steps for the good substrates benzylpenicillin and phenoxymethylpenicillin; this depends on determining the fraction of enzyme that is present as acyl-enzyme in the steady state.     

The pH-dependence of sugar-transport and glycolysis in cultured Ehrlich ascites-tumour cells.

1. pH-dependence of glycolysis has generally been ascribed to the effects of pH on the activities of glycolytic enzymes. The present study shows that sugar transport is pH-dependent in cultured Ehrlich ascites-tumour cells. 2. The rates of glucose consumption, of 3-O-methylglucose transport, and of 2-deoxyglucose transport and phosphorylation increased as linear functions of pH, as the pH of the cell culture medium was increased from 6.1 to 8.5. Transport of glucose, as measured in ATP-depleted cells, was pH-dependent to the same extent as transport of the non-metabolizable sugars. 3. Glucose consumption rates were about 8-fold higher at pH 8.5 than at pH 6.4. About 65-85% of glucose was converted into lactate. Sugar transport rates were 2.5-fold higher at pH 8.5 than at pH 6.3. 4. pH affected both simple diffusion and facilitated diffusion. pH effect was mainly on the Vmax. of 2-deoxyglucose uptake, and on the rapid-uptake phase of 3-O-methylglucose transport. 5. It was estimated that about 70% of the pH effect on the rates of glucose consumption may be due to the effect on sugar transport and the remainder to the effect on the activities of glycolytic enzymes.     

Sulfhydryl group modification of sarcoplasmic reticulum membranes.

Modification of calcium-translocating sarcoplasmic reticulum membranes (SR) with 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) reveals four classes (kinetic sets) of sulfhydryl groups. Of the 25 mol/1.5 X 10(5) G OF SR protein (i.e., containing 1 mol of ATPase protein) estimated in the presence of sodium dodecyl sulfate, 8 mol are unreactive, while 7, 8, and 2 mol display pseudo-first-order rate constants (k1) of 0.16, 0.68, and 8.3 min(-1), respectively (25 decrees C, pH 7.8, 4 MM Nbs2). Under these conditions, the Ca-ATPase activity is lost with k1 = 0.73 min(-1), whereas the Ca-independent ATPase activity is essentially unchanged. These results are little changed by the presence of Mg2+ or Ba2+ in the modification mixture, while Ca2+ or Sr2+ causes all 16-17 reactable sulfhydryls to be modified with k1 = 0.50 and 0.53 min(-1), respectively. The corresponding values for the loss of Ca-ATPase activity are 0.53 and 0.67 min(-1); this suggests that blocking of only one of the 16-17 SH groups inactivates the enzyme, i.e., that there is a single "essential" SH group. The midpoint of the transition between the Ca2+-free and Ca2+-modification patterns occurs at a free Ca2+ concentration of about 0.9 muM, implying that it is Ca2+ binding at the active sites (KD = 0.1 muM), rather than at the low-affinity nonspecific sites, that effects a conformation change in the ATPase protein (which contains greater than 90% of the cysteines). A calcium-induced conformation change is also suggested by increased ultraviolet absorbance spectrum of the purified ATPase protein upon calcium binding. If protein-lipid interaction is disrupted with deoxycholate or Triton X-100 (which does not destroy the Ca-ATPase activity and hence presumably leaves the tertiary structure of the ATPase protein largely intact), 95% of the sulfhydryls react with Nbs2 considerably faster; thus, at 2 mg/ml o- deoxycholate, 14 groups react with k1 greater than 20, 5 with k1 = 2.3, and 5 with k1 = 0.4 min(-1). These results suggest that the inaccessibility of SH groups in the absence of detergents is due to extensive interaction of the bilayer phospholipids with the ATPase protein.     

Kinetics of inactivation of beta-lactamase I by 6 beta-bromopenicillanic acid.

The kinetics of the inactivation of beta-lactamase I from Bacillus cereus 569 by preparations of 6 alpha-bromopenicillanic acid showed unexpected features. These can be quantitatively accounted for on the basis of the inactivator being the epimer, 6 beta-bromopenicillanic acid. At pH 9.2, the rate-determining step in the inactivation is the formation of the inactivator. When pure 6 beta-bromopenicillanic acid is used to inactivate beta-lactamase I, simple second-order kinetics are observed. The inactivated enzyme has a new absorption peak at 326 nm. The rate constant for inactivation has the same value as the rate constant for appearance of absorption at 326 nm; the rate-determining step may thus be fission of the beta-lactam ring of 6 beta-bromopenicillanic acid. Inactivation is slower in the presence of substrate, and the observed kinetics can be quantitatively accounted for on a simple competitive model. The results strongly suggest that inactivation is a consequence of reaction at the active site.     

Substrate-induced deactivation of penicillinases. Studies of beta-lactamase I by hydrogen exchange.

The conformational motility of beta-lactamase I from Bacillus cereus was studied by hydrogen exhange. The time course of the isotopic replacement of peptide hydrogen atoms was followed by 'exchange-in' or 'exchange-out' experiments. Many of the substrates for this enzyme that have o-substituted aromatic or heterocyclic side chains (e.g. methicillin or cloxacillin) are known to effect a decrease in enzymic activity ('substrate-induced deactivation'). There was a marked discontinuity in the exchange-out curve when methicillin or cloxacillin was diffused into the enzyme solution. About one-half of the hydrogen atoms that were probed were affected by the presence of these substrates, and the change in the reactivity of the hydrogen atoms was also large. Substrates that do not bring about deactivation (benzylpenicillin and cephalosporin C) do not affect the hydrogen exchange, nor do reversible competitive inhibitors such as the penicilloic acid or penilloic acid. On the other hand, Zn2+ ions do affect the hydrogen exchange; their effect is similar to that of methicillin or cloxacillin.     

Maleylation and pH-dependence of Streptomyces griseus protease B.

The enzymatic activity of Streptomyces griseus protease B (SGPB) was measured over pH range 8.4--11.5 using a specific new, chromophoric substrate N-succinyl-glycyl-glycyl-L-phenylalanine p-nitroanilide. It was found that the activity is dependent on ionization of a single group with apparent pK = 10.84, possibly lysine-125. Maleylation of the epsilon-amino group of this lysine was linearily associated with the loss of enzymatic activity. It is therefore suggested that the electrostatic interaction between the side chain of lysine-125 and the alpha-carboxyl group of the C-terminal tyrosine is crucial to the active conformation of the enzyme. In contrast the maleylation of the alpha-amino group of the N-terminal isoleucine was rapid but could not be correlated to the loss of activity.