Critical ionizing groups in Aeromonas neutral protease.

Aeromonas neutral protease possesses two residues critical to its activity. One has a pKa of 5.5 in both the free enzyme and the enzyme-substrate complex and must be deprotonated for maximal activity. The other, which ionizes at pH 7.1 in the free enzyme and at pH 7.4 in the enzyme-substrate complex, must be protonated for optimal enzyme action. The protease is reversibly inhibited by aminoacyl hydroxamates, peptides containing a phenylalanyl residue, phosphoryl-L-phenylalanylglycylglycine, and by beta-phenylpropionyl-L-phenylalanine. The pH dependence of inhibition by the latter revealed that a residue with a pKa of 5.6 influences inhibitor binding. Compounds containing both a hydroxamido group and a chloroacetyl group are particularly effective in inactivating the enzyme, and inhibition is enhanced by hydrophobic residues. Thus, a 33-fold molar excess of chloroacetyl-N-hydroxy-L-phenylalanyl-L-alanyl-L-alanine amide rapidly inactivated Aeromonas neutral protease. Carbethoxylation experiments resulted in a 90% loss in activity which was fully reversible by hydroxylamine; spectral analysis indicated the involvement of a single histidine residue. Protection against both esterification and carbethoxylation was furnished by the presence of beta-phenylproprionyl-L-phenylalanine. Inactivation experiments suggest that a glutamic or aspartic acid and a histidine residue are responsible for the pKa values revealed by pH dependence studies.

Hydroxamate-induced spectral perturbations of cobalt Aeromonas aminopeptidase.

The absorption spectrum of cobalt(II)-substituted Aeromonas aminopeptidase is markedly perturbed by the presence of equimolar concentrations of D-amino acid hydroxamates and acyl hydroxamates that have previously been shown to be powerful inhibitors of this enzyme (Wilkes, S. H., and Prescott, J. M. (1983) J. Biol. Chem. 258, 13517-13521). D-Valine hydroxamate produces the most distinctive perturbation, splitting the characteristic 527 nm absorption peak of the cobalt enzyme to form peaks at 564, 520, and 487 nm with molar extinction values of 126, 98, and 67 M-1 cm-1, respectively. A qualitatively similar perturbation, albeit with lower extinction values, results from the addition of D-leucine hydroxamate, whereas D-alanine hydroxamate perturbs the spectrum, but does not evoke the peak at 564 nm. In contrast, hydroxamates of L-valine and L-leucine in concentrations equi-molar to that of the enzyme produce only faint indications of change in the spectrum, but the hydroxamates of several other L-amino acids perturb the spectrum essentially independently of the identity of the side chain and in a qualitatively different manner from that of D-valine hydroxamate and D-leucine hydroxamate. At the high enzyme:substrate ratios used in the spectral experiments, L-leucine hydroxamate and L-valine hydroxamate proved to be rapidly hydrolyzed, hence their inability to perturb the spectrum of the cobalt-substituted enzyme during the time course of a spectral experiment. Values of kcat for L-amino acid hydroxamates, all of which are good reversible inhibitors of the hydrolysis of L-leucine-p-nitroanilide by Aeromonas aminopeptidase, were found to range from 0.01 min-1 to 5.6 min-1 for the native enzyme and from 0.27 min-1 to 108 min-1 for the cobalt-substituted enzyme; their km values toward the cobalt aminopeptidase range from 1.2 X 10(-7) M to 1.9 X 10(-5) M. The mutual exclusivity of binding for hydroxamate inhibitors and 1-butaneboronic acid, previously shown by kinetics (Baker, J. O., Wilkes, S. H., Bayliss, M. E., and Prescott, J. M. (1983) Biochemistry 22, 2098-2103), was reflected in the characteristic spectra produced by these two types of inhibitors.

Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates.

Aeromonas aminopeptidase contains two nonidentical metal binding sites that have been shown by both spectroscopy and kinetics to be capable of interacting with one another [Prescott, J.M., Wagner, F.W., Holmquist, B., & Vallee, B.L. (1985) Biochemistry 24, 5350-5356]. The effects of metal ion substitutions on the susceptibility of the p-nitroanilides of L-alanine, L-valine, and L-leucine--substrates that are hydrolyzed at widely differing rates by native Aeromonas aminopeptidase--were studied by determining values of kcat and Km for the 16 metalloenzymes that result from all possible combinations of Zn2+, Co2+, Ni2+, and Cu2+ in each of the two sites. The different combinations of metal ions and substrates yield a broad range in kinetic values; kcat varies by more than 1800-fold, Km by 3000-fold, and kcat/Km ratios by more than 10,000. L-Leucine-p-nitroanilide is by far the most susceptible of the three substrates, and the hyperactivation previously observed with aminopeptidase containing either Ni2+ or Cu2+ in the first binding site and Zn2+ in the second site occurs only with the two poorer substrates, L-alanine-p-nitroanilide and L-valine-p-nitroanilide. Although the enzyme with Zn2+ in both sites hydrolyzes the substrates with N-terminal alanine and valine poorly, it is extremely effective toward L-leucine-p-nitroanilide. Neither metal binding site can be identified as controlling either Km or kcat; both parameters are influenced by the identity of the metal ions, by the site each occupies, and, most strongly, by the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

The slow, tight binding of bestatin and amastatin to aminopeptidases.

Bestatin reversibly inhibits Aeromonas aminopeptidase (EC 3.4.11.10) in a process that is remarkable for its unusual degree of time dependence. The binding of bestatin by both Aeromonas aminopeptidase and cytosolic leucine aminopeptidase (EC 3.4.11.1) is slow and tight, with Ki values (determined from rate constants) of 1.8 X 10(-8) and 5.8 X 10(-10) M, respectively. In contrast, microsomal aminopeptidase (EC 3.4.11.2) binds bestatin in a rapidly reversible process with a Ki value of 1.4 X 10(-6) M. Kinetic analysis of the slow inhibition observed is facilitated by the use of a variety of experimental treatments, primarily measurements made during pre-equilibrium; however, careful selection of conditions permits use also of steady state observations. When titrated with bestatin, 1 mol of cytosolic leucine aminopeptidase (containing 6 g atoms each of zinc and manganese) is rendered 80% inactive by 1 mol of inhibitor, thus suggesting that enzymatic activity depends on one active site/hexamer; titration of Aeromonas aminopeptidase by bestatin reveals a 1:1 stoichiometry. Amastatin inhibits all three aminopeptidases through the mechanism of slow, tight binding with Ki values ranging from 3.0 X 10(-8) to 2.5 X 10(-10) M. This behavior of microsomal aminopeptidase contrasts sharply with its rapidly reversible inhibition by bestatin. The slow, tight binding observed with five of the six aminopeptidase-inhibitor pairs investigated suggests the formation of a transition state analog complex between the enzyme and inhibitor. Physical evidence consistent with this possibility was provided by the observation that both bestatin and amastatin perturb the absorption spectrum of cobalt Aeromonas aminopeptidase.

Spectral and kinetic studies of metal-substituted Aeromonas aminopeptidase: nonidentical, interacting metal-binding sites.

Apoenzyme prepared by removal of the 2 mol of Zn2+/mol from Aeromonas aminopeptidase is inactive. Addition of Zn2+ reactivates it completely, and reconstitution with Co2+, Ni2+, or Cu2+ results in a 5.0-, 9.8-, and 10-fold more active enzyme than native aminopeptidase, respectively. Equilibrium dialysis and spectral titration experiments with Co2+ confirm the stoichiometry of 2 mol of metal/mol. The addition of only 1 mol of metal/mol completely restores activity characteristic of the particular metal. Interaction between the two sites, however, causes hyperactivation; thus, addition of 1 mol of Zn2+/mol subsequent to 1 mol of Co2+, Ni2+, or Cu2+ per mole increases activity 3.2-, 42-, or 59-fold, respectively. The cobalt absorption spectrum has a peak of 527 nm with a molar absorptivity of 53 M-1 cm-1 for 1 mol of cobalt/mol, which increases to 82 M-1 cm-1 for a second cobalt atom and is unchanged by further addition of Co2+. Circular dichroic (CD) and magnetic CD spectra indicate that the first Co2+ binding site is tetrahedral-like and that the second is octahedral-like. Stoichiometric quantities of 1-butylboronic acid, a transition-state analogue inhibitor of the enzyme [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322], profoundly affects absorption, CD, and MCD spectra, but n-valeramide, a substrate analogue inhibitor, has no effect. These findings suggest that the tetrahedral-like site is catalytic and the other octahedral-like site is regulatory or structural.

A transition-state-analog inhibitor influences zinc-binding by Aeromonas aminopeptidase.

The transition-state-analog inhibitor, 1-butaneboronic acid, markedly enhances the uptake of one g-atom of Zn2+ ions from a metal ion buffer system by Zn-depleted Aeromonas aminopeptidase. In contrast, a substrate-analog inhibitor, n-valeramide, does not perturb the equilibrium between Zn2+ ions and the enzyme in a metal ion buffer system. These results establish a role for metal ions in the binding of 1-butaneboronic acid to Aeromonas amino-peptidase and strongly imply that a bound Zn2+ ion interacts directly with substrate during catalysis but not during initial binding of substrate.

Stereospecificity of amino acid hydroxamate inhibition of aminopeptidases.

Hydroxamates of amino acids and aliphatic acids are effective inhibitors of Aeromonas proteolytica amino-peptidase (EC 3.4.11.10) and of both the cytosolic (EC 3.4.11.1) and microsomal (EC 3.4.11.2) aminopeptidases of swine kidney. Cytosolic leucine aminopeptidase and the Aeromonas enzyme were inhibited to a greater extent by D isomers than by the L enantiomorphs, manganese-activated kidney cytosolic leucine aminopeptidase being inhibited 10 times more effectively by D-leucine and D-valine hydroxamic acids than by the L isomers. The D isomers of these two compounds inhibited Aeromonas aminopeptidase to an even greater extent with Ki values of 2 X 10(-9) and 5 X 10(-9), respectively, whereas the corresponding L isomers were bound 150 times less tightly. With the Aeromonas enzyme, a comparison of inhibition by racemic mixtures with that of the corresponding L isomers indicated that in all cases the contribution of the D isomer was predominant. Isocaproic hydroxamic acid inhibited this enzyme equally well as L-leucine hydroxamic acid, indicating that the amino group orientation in the D isomer contributes to the binding efficacy. Swine kidney microsomal aminopeptidase was also inhibited by D isomers of leucine and valine hydroxamic acids but in contrast to the other two enzymes, the inhibition was 10-fold less than that observed for the corresponding L isomers. Cytosolic leucine aminopeptidase with either 6 g atoms of zinc per mol or 12 g atoms of zinc per mol was inhibited only slightly by any of the hydroxamic acid compounds; evidently enzyme-bound manganese (or magnesium) is specific for hydroxamate binding to this aminopeptidase.

One hundred fold increased activity of Aeromonas aminopeptidase by sequential substitutions with Ni(II) or Cu(II) followed by zinc.

Full substitution of Cu(II) or Ni(II) for the two g-atom zinc in Aeromonas aminopeptidase hyperactivates the enzyme 6.5 and 25 fold respectively. Even greater enhancements of activity can be achieved with mixed metal substitutions. Thus, apoenzyme reactivated by first adding one g-atom zinc followed by one g-atom of either Cu(II) or Ni(II) is 15 and 22 times more active than the native enzyme. Reversing the order, i.e. by first adding either one g-atom Cu(II) or Ni(II) followed by one g-atom zinc, activates the enzyme nearly 100 fold. The order of metal addition is critical and suggests the existence of two non-identical metal sites, each with a different function.

ES complexes of Aeromonas aminopeptidase: direct observation by stopped-flow fluorescence.

Intermediates of Aeromonas aminopeptidase are monitored through fluorescence generated by radiationless energy transfer (RET) between enzyme tryptophans and the dansyl group of the bound substrate. Upon binding of the substrate enzyme tryptophan fluorescence is quenched and substrate dansyl fluorescence enhanced. These processes are reversed upon hydrolysis of the Leu-Ala bond and release of Ala-DED from the enzyme. Stopped-flow RET kinetic analysis yields values of kcat = 36 sec-1 and Km = 3.7 microM at pH 7.5 and 20 degrees C. These values represent the highest kcat/Km ratio, 1 X 10(7) M-1 sec-1, of any substrate for Aeromonas aminopeptidase. The excellent binding properties of the peptide permit direct visualization of ES complexes even at enzyme concentrations of 10(-7) M.

Isolation and some characteristics of glutathione reductase from rabbit erythrocytes (38548).

Glutathione reductase from rabbit erythrocytes was pruified to homogeneity and found to be a monomer with a mol wt of 60,000. Both NADPH and HADH were capable of acting as cofactors for the reduction of GSSG and the following kinetic values were obtained: Km, GSSG = 120 muM; Km, NADPH = 37 muM; Vmax = 23 mumoles NADPH/min/mg protein, Km, NADH = 420 muM; Vmax = 3 mumoles NADH/min/mg protein. Rabbit erythrocyte GR exhibited substrate inhibition, and was susceptible to inhibition by p-hydroxymercuribenzoate under certain conditions.