Nonhydrolyzable Substrate Analog Research Articles

Probing the renin active site by collisional quenching of endogenous fluorescence.

The structural and enzymatic aspects of renin are of great interest in hypertension research. In this paper, we examine the solution accessibility of the three tryptophan (Trp) residues of mouse submaxillary gland renin by solute collisional fluorescence quenching. Our studies indicate that there are two "classes" of Trp residues in renin: class I, a class of Trp residues which are at or near the surface of renin and fully accessible to the fluorescence quencher iodide; and class II, a class of Trp residues which are, for practical experimental conditions, totally inaccessible to the aqueous solution. The former class contains 2 Trp residues, while only a single Trp is identified in the latter class. The presence of a tetradecapeptide substrate or a nonhydrolyzable substrate analogue (peptide H-77) lowers the accessibility of iodide to the class I Trp residues. These data indicate that the class I Trp residues are at or near the peptide-binding site of renin. In addition, the finding that the class I Trp residues are quantitatively quenched more efficiently than the Trp model compound indole suggests that the environment of the class I tryptophans may be positively charged, and thus have a higher "local" concentration of iodide. These data, taken together with the available sequence and computer-generated three-dimensional structure of renin, permit us to speculate that the class I Trp residues are Trp-39 and Trp-300. This solution study of renin structure is discussed in light of the known information about renin catalysis and physiology.

Complex photophysics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex.

The fluorescence emission of the single tryptophan in porcine pancreatic phospholipase A2, its zymogen, and a micellar complex of the enzyme with the nonhydrolyzable substrate analogue n-hexadecylphosphocholine has been studied by both steady-state and time-resolved techniques. Stern-Volmer quenching studies with acrylamide indicate that, both in the enzyme and in the zymogen, the tryptophan is exposed to solvent. Similar studies with ionic quenchers show that there is appreciable ionic character to the tryptophan environment. Single photon counting fluorescence measurements were performed using a high repetition rate synchronously pumped dye laser as a light source. When tryptophan fluorescence is collected with a broad-band (80-nm) emission filter, the decay kinetics in the enzyme and the zymogen require at least three, and often four, exponential terms for a proper description. The decay kinetics can be adequately described by three exponential terms when the fluorescence is collected at specific wavelengths by using narrow (10-nm) band-pass filters. The lifetimes are approximately constant across the emission band, but the amplitudes vary with the fraction of the long lifetime increasing at longer emission wavelengths. Formation of a complex between phospholipase A2 and micelles of n-hexadecylphospohocholine produces large changes in the tryptophan emission that are associated with transfer to a hydrophobic environment. The decay kinetics of tryptophan in the enzyme/micelle complex appears to require only two exponential terms. This is the first reported instance of fluorescence data from a single tryptophan protein requiring more than double-exponential decay kinetics. The results are discussed in terms of the range of environments sampled by the tryptophan residue and the resulting distribution of lifetimes.

Des-1-25-fructose-1,6-bisphosphatase, a nonallosteric derivative produced by trypsin treatment of the native protein.

Limited tryptic digestion of pig kidney fructose-1,6-bisphosphatase in the presence of magnesium ions results in the formation of an active enzyme derivative which is no longer inhibited by the allosteric effector AMP. The presence of AMP during incubation of fructose-1,6-bisphosphatase with trypsin protects against the loss of AMP inhibition. By contrast, the presence of the nonhydrolyzable substrate analog fructose 2,6-bisphosphate accelerates the rate of formation of that form of fructose-1,6-bisphosphatase which is insensitive to AMP inhibition. Sodium dodecyl sulfate-polyacrylamide electrophoresis of samples taken during trypsin treatment shows that the loss of AMP inhibition parallels the conversion of the native 36,500 molecular weight fructose-1,6-bisphosphatase subunit into a 34,000 molecular weight species. Automated Edman degradation of trypsin-treated fructose-1,6-bisphosphatase following gel filtration shows a single sequence beginning at Gly-26 in the original enzyme, but no changes in the COOH-terminal region of fructose-1,6-bisphosphatase. Thus, the proteolytic product has been characterized as "des-1-25-fructose-1,6-bisphosphatase." A comparison of the kinetic properties of control enzyme and des-1-25-fructose-1,6-bisphosphatase reveals some differences in properties (pH optimum, Ka for Mg2+, K+ activation, inhibition by fructose 2,6-bisphosphate) between the two enzymes, but none is so striking as the complete loss of AMP sensitivity shown by des-1-25-fructose-1,6-bisphosphatase. The loss of AMP inhibition is due to the loss of AMP-binding capacity, but it is not known at this stage whether residues of the AMP site are present in the 25-amino acid NH2-terminal region or the removal of this region leads to a conformational change that abolishes the function of an AMP site located elsewhere in the molecule.

Affinity labeling of the (Na + + Mg 2+)-ATPase from A choleplasma laidlawii B membranes by the 2′,3′-dialdehyde derivative of adenosine 5′-triphosphate

The (Na + + Mg 2+)-ATPase of the Acholeplasma laidlawii B plasma membrane was inactivated by the 2′,3′-dialdehyde derivative of ATP (oATP). oATP behaved as a reversible competitive inhibitor of this ATPase and was slowly hydrolyzed by the enzyme. In addition, oATP induced an irreversible inactivation of the enzyme. A 62% inactivation of the enzyme correlated with the binding of 16 moles of oATP per mole of the enzyme. In the presence of 5′-adenylyl imidodiphosphate, a non-hydrolyzable substrate analogue, the stoichiometry was 8 moles oATP per mole of ATPase. By SDS-polyacrylamide gel electrophoresis, [U- 14C]oATP was found to bind covalently to four of the five subunits of the enzyme, but specific labeling was highest for the γ-subunit of the ATPase.

Properties of F1-ATPase from the uncD412 mutant of Escherichia coli

Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate. In most properties that were studied, uncD412 F1-ATPase resembled normal E. coli F1-ATPase. Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites. Binding of the non-hydrolysable substrate analogue adenosine 5'-[beta gamma-imido]triphosphate (p[NH]ppA) to the three exchangeable sites showed apparent negative co-operativity. The binding affinities for p[NH]ppA, and also ADP, at the exchangeable sites were similar in the two enzymes. Both enzymes were inhibited by efrapeptin, aurovertin and p[NH]ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5'-adenosine. Km values for CaATP and MgATP were similar in the two enzymes. uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity. The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies. However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for 'promotion' of catalysis in this enzyme.

The interaction of dialkyl ether lecithins with phospholipase A2 (Naja naja naja).

Dialkyl lecithins are nonhydrolyzable substrate analogues for phospholipase A2 (Naja naja naja). Short chain dialkyl lecithins (which form monomers and micelles), and long chain ether-linked lecithins (which form bilayers or can be solubilized in detergent micelles) have been used to study phospholipase A2/phospholipid interactions. The results of gel filtration, 1H and 31P NMR spectroscopy, kinetic assays and UV difference spectroscopy show that binding of the ether-linked lecithins is independent of metal ion in pure lecithin systems (monomer, micelle, or bilayer), but requires Ca2+ for tight binding when Triton X-100 is used to solubilize the lecithin in mixed micelles. The apparent KD for dihexadecyl phosphatidylcholine is 2 mM in sonicated vesicles, but is increased to 6 mM in Triton-mixed micelles in the presence of Ca2+. When phospholipase A2 binds monomeric and micellar dialkyl lecithin, the lecithin phosphate resonance is broadened considerably, indicating that head group motion is restricted. Ca2+ enhances this interaction with the head group in these pure lipid systems. In similar 31P NMR experiments with dihexadecyl phosphatidylcholine solubilized in Triton X-100 mixed micelles, phospholipase A2 does not cause comparable broadening of the phosphate resonance, even in the presence of Ca2+ where gel filtration shows binding occurs. The kinetic effect of the ether-linked lipid inhibitors on phospholipase activity toward ester-linked lecithins is also modulated by the presence of Triton X-100. Ether-linked lecithins are much more potent inhibitors in two-component (dialkyl- diacyl-) lecithin micelles and bilayers than in complex mixed micelles with Triton X-100. Detergent-induced changes in the characteristics of the micellar interface are implicated in this inhibition phenomenon. The interaction of phospholipase A2 with sonicated vesicles of dihexadecyl phosphatidylcholine has also been examined by 1H and 31P NMR spectroscopy. When phospholipase A2 binds to vesicles, it promotes loss of encapsulated lanthanide ions, and appears to accelerate transbilayer phospholipid "flip-flop" rates. These results are discussed in terms of membrane asymmetry studies using phospholipase A2.

Kinetics and optical spectroscopic studies on the purple acid phosphatase from beef spleen.

A new purification scheme has been developed for the purple acid phosphatase from beef spleen; typical yields are 8 mg of homogeneous enzyme per kg of spleen in only five steps. Kinetics studies have shown that the enzyme is strongly inhibited by fluoride, phosphate, and [p-(acetylamino)-benzyl]phosphonate, a nonhydrolyzable substrate analogue; the last two of these show simple competitive inhibition. In contrast, cyanide, azide, tartrate, and p-nitrophenol show no inhibition at concentrations up to 10 mM. Molecular weight estimations by gel electrophoresis and gel permeation chromatography give a value of 40 000 for the native enzyme, which is shown to consist of two subunits of apparent molecular weight 24 000 and 15 000. Careful metal analyses indicate the presence of 2.1 +/- 0.1 iron atoms per enzyme molecule, and less than 0.1 copper, zinc, nickel, or manganese atom per enzyme. The purple enzyme (lambda max 550 nm) is reversibly converted to a pink, active form (lambda max 505 nm) upon treatment with mild reducing agents (dithioerythritol or ascorbate). Addition of competitive inhibitors to the pink form causes rapid reversion to the purple form. Electron paramagnetic resonance spectroscopy at several temperatures showed only weak g = 4.3 signals (less than 0.1 spin/molecule) for the native, reduced, and inhibited forms of the enzyme.

Studies on nucleosides and nucleotides. LXXXVIII. Synthesis of a non-hydrolyzable substrate analog of ribonuclease Tl, 2'-deoxy-2'-fluoroguanylyl-(3'-5')-uridine.

2'-Deoxy-2'-fluoroguanylyl-(3'-5')-uridine (GfpU) was synthesized as a non-hydrolyzable substrate analog of ribonuclease T1. 2'-Deoxy-2'-fluoroguanosine was derivatized to the N2-isobutyryl-5'-monomethoxytrityl compound and condensed with 2', 3'-di-O-acetyluridine 5'-monophosphate by the use of dicyclohexylcarbodiimide to obtain GfpU. For structural elucidation of the dinucleoside monophosphate, 2'-deoxy-2'-fluoroguanosine 3'-monophosphate was also synthesized. The conformation of GfpU was found to take a right-handed, weakly stacked form as deduced by ultraviolet, circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy.