Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK(a) of the inhibitor carboxylate group is lowered by at least 1.5 pK(a) units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-(13)C]-L-tryptophan is ≤3.71? from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-(13)C]-L-tryptophan is not bound in the S(2)' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.     

A new lysine derived glyoxal inhibitor of trypsin,its properties and utilization for studying the stabilization of tetrahedral adducts by trypsin

New trypsin inhibitors Z-Lys-COCHO and Z-Lys-H have been synthesised. K_i values for Z-Lys-COCHO, Z-Lys-COOH, Z-Lys-H and Z-Arg-COOH have been determined. The glyoxal group (–COCHO) of Z-Lys-COCHO increases binding ~300 fold compared to Z-Lys-H. The α-carboxylate of Z-Lys-COOH has no significant effect on inhibitor binding. Z-Arg-COOH is shown to bind ~2 times more tightly than Z-Lys-COOH. Both Z-Lys-¹³COCHO and Z-Lys-CO¹³CHO have been synthesized. Using Z-Lys-¹³COCHO we have observed a signal at 107.4 ppm by ¹³C NMR which is assigned to a terahedral adduct formed between the hydroxyl group of the catalytic serine residue and the ¹³C-enriched keto-carbon of the inhibitor glyoxal group. Z-Lys-CO¹³CHO has been used to show that in this tetrahedral adduct the glyoxal aldehyde carbon is not hydrated and has a chemical shift of 205.3 ppm. Hemiketal stabilization is similar for trypsin, chymotrypsin and subtilisin Carlsberg. For trypsin hemiketal formation is optimal at pH 7.2 but decreases at pHs 5.0 and 10.3. The effective molarity of the active site serine hydroxyl group of trypsin is shown to be 25300 M. At pH 10.3 the free glyoxal inhibitor rapidly (t_1/2=0.15 h) forms a Schiff base while at pH 7 Schiff base formation is much slower (t_1/2=23 h). Subsequently a free enol species is formed which breaks down to form an alcohol product. These reactions are prevented in the presence of trypsin and when the inhibitor is bound to trypsin it undergoes an internal Cannizzaro reaction via a C2 to C1 alkyl shift producing an α-hydroxycarboxylic acid.     

Determination of the structure of tetrahedral transition state analogues bound at the active site of chymotrypsin using 18O and 2H isotope shifts in the 13C NMR spectra of glyoxal inhibitors

The peptide-derived glyoxal inhibitor Z-Ala-Pro-Phe-glyoxal, where Z is benzyloxycarbonyl, is an extremely potent inhibitor of chymotrypsin. When it is bound to chymotrypsin both the glyoxal (RCOCHO) keto and aldehyde carbons are sp3 hybridized with chemical shifts of 100.7 and 91.4 ppm, respectively. However it is has not been shown whether these carbons are bound as hydrates or whether the active-site serine has reacted with them to form the corresponding hemiketal or hemiacetal. In this study we use 18O isotope shifts to determine whether one or two exchangeable oxygen atoms are attached to the glyoxal keto or aldehyde carbons when it is free in water or bound to alpha-chymotrypsin. Both the 18O isotope shifts at the free and enzyme-bound aldehyde carbons were approximately 0.04 ppm showing that it is hydrated in both the free and bound forms. The 18O isotope shift for the free hydrated keto carbon at 96.6 ppm was 0.046-0.049 ppm, but this was reduced to 0.026 ppm when the glyoxal inhibitor was bound to alpha-chymotrypsin showing that the nonexchangeable serine hydroxyl group has formed a hemiketal with glyoxal keto carbon. Deuterium isotope shifts on the 13C NMR signals from the glyoxal inhibitor when it free and hydrated, when it is bound to chymotrypsin, as well as when it forms a model hemiketal confirm that the serine hydroxyl group has formed a hemiketal with the glyoxal keto carbon. The reasons for the different reaction specificities of glyoxal inhibitors for the active-site nucleophiles of serine and cysteine proteases are discussed.     

NMR study of the inhibition of pepsin by glyoxal inhibitors: mechanism of tetrahedral intermediate stabilization by the aspartyl proteases

Z-Ala-Ala-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of pepsin with a Ki = 89 +/- 24 nM at pH 2.0 and 25 degrees C. Both the ketone carbon (R13COCHO) and the aldehyde carbon (RCO13CHO) of the glyoxal group of Z-Ala-Ala-Phe-glyoxal have been 13C-enriched. Using 13C NMR, it has been shown that when the inhibitor is bound to pepsin, the glyoxal keto and aldehyde carbons give signals at 98.8 and 90.9 ppm, respectively. This demonstrates that pepsin binds and preferentially stabilizes the fully hydrated form of the glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal. From 13C NMR pH studies with glyoxal inhibitor, we obtain no evidence for its hemiketal or hemiacetal hydroxyl groups ionizing to give oxyanions. We conclude that if an oxyanion is formed its pKa must be >8.0. Using 1H NMR, we observe four hydrogen bonds in free pepsin and in pepsin/Z-Ala-Ala-Phe-glyoxal complexes. In the pepsin/pepstatin complex an additional hydrogen bond is formed. We examine the effect of pH on hydrogen bond formation, but we do not find any evidence for low-barrier hydrogen bond formation in the inhibitor complexes. We conclude that the primary role of hydrogen bonding to catalytic tetrahedral intermediates in the aspartyl proteases is to correctly orientate the tetrahedral intermediate for catalysis.     

Ionisations within a subtilisin-glyoxal inhibitor complex

Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of subtilisin with a K(i)=2.3+/-0.2 microM at pH 7.0 and 25 degrees C. Using Z-Ala-Pro-[2-(13)C]Phe-glyoxal we have detected a signal at 107.3 ppm by (13)C NMR, which we assign to the tetrahedral adduct formed between the hydroxy group of serine-195 and the (13)C-enriched keto-carbon of the inhibitor. The chemical shift of this signal is pH independent from pH 4.2 to 7.0 and we conclude that the oxyanion pK(a)<3. This is the first observation of oxyanion formation in a reversible subtilisin-inhibitor complex. The inhibitor is bound as a hemiketal which is in slow exchange with the free inhibitor. Inhibitor binding depends on a pK(a) of approximately 6.5 in the free enzyme and on a pK(a)<3.0 when the inhibitor is bound to subtilisin. Protonation of the oxyanion promotes the disassociation of the inhibitor. We show that oxyanion formation cannot be rate limiting during catalysis and that subtilisin stabilises the oxyanion by at least 45.1 kJ mol(-1). We conclude that if the energy required for oxyanion stabilisation is utilised as binding energy in drug design it should make a significant contribution to inhibitor potency.     

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK_a of the inhibitor carboxylate group is lowered by at least 1.5 pK_a units when it binds to either enzyme. The signal at ~ 183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-¹³C]-L-tryptophan is ≤ 3.71 Å from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-¹³C]-L-tryptophan is not bound in the S₂′ subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~ 183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.     

Effects of advanced glycation end products-inductor glyoxal and hydrogen peroxide as oxidative stress factors on rat retinal organ cultures and neuroprotection by UK-14,304

Retinal ganglion cell degeneration is supposed to be mediated by reactive oxygen species (ROS) and advanced glycation end products (AGEs). The alpha2-adrenergic agonist, 5-bromo- N -(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (brimonidine; UK-14,304), is said to exert a neuroprotective effect. To investigate these mechanisms in detail, we exposed rat whole mounts to glyoxal or H₂O₂ and treated them with either UK-14,304 alone or additionally with the phosphatidylinositide 3 kinase (PI3) kinase inhibitor, 2-(4-Morpholinyl)-8-phenyl-4 H -1-benzopyran-4-one (Ly 294002). The accumulation of Nε-[carboxymethyl] lysine (CML) was assessed immunohistochemically and changes in intracellular pH (pHi), mitochondrial transmembrane potential (MTMP) and ROS production in cell bodies of multipolar ganglion cell layer were studied by intravital fluorescence microscopy and confocal laser scanning microscopy. Ultrastructural changes in mitochondria of multipolar ganglion cell layer cell bodies were determined by transmission electron microscopy. We found that glyoxal and H₂O₂ increased accumulation of CML-modified proteins and ROS production and decreased pHi and MTMP in cell bodies of multipolar ganglion cell layer. UK-14,304 could prevent production of ROS, accumulation of CML-modified proteins, ameliorate acidification, preserve MTMP and attenuate ultrastructural damages of ganglion cell mitochondria. Ly 294002 reversed the UK-14,304-mediated attenuation of CML and ROS production. We conclude that the protective effects of UK-14,304 seem partly to be mediated by PI3 kinase-dependent pathways.     

X-ray structure of human stromelysin catalytic domain complexed with nonpeptide inhibitors: implications for inhibitor selectivity.

Effective inhibitors of matrix metalloproteinases (MMPs), a family of connective tissue-degrading enzymes, could be useful for the treatment of diseases such as cancer, multiple sclerosis, and arthritis. Many of the known MMP inhibitors are derived from peptide substrates, with high potency in vitro but little selectivity among MMPs and poor bioavailability. We have discovered nonpeptidic MMP inhibitors with improved properties, and report here the crystal structures of human stromelysin-1 catalytic domain (SCD) complexed with four of these inhibitors. The structures were determined and refined at resolutions ranging from 1.64 to 2.0 A. Each inhibitor binds in the active site of SCD such that a bulky diphenyl piperidine moiety penetrates a deep, predominantly hydrophobic S'1 pocket. The active site structure of the SCD is similar in all four inhibitor complexes, but differs substantially from the peptide hydroxamate complex, which has a smaller side chain bound in the S'1 pocket. The largest differences occur in the loop forming the "top" of this pocket. The occupation of these nonpeptidic inhibitors in the S'1 pocket provides a structural basis to explain their selectivity among MMPs. An analysis of the unique binding mode predicts structural modifications to design improved MMP inhibitors.     

A single step purification for autolytic zinc proteinases

We describe a novel single-step method for the purification of stromelysin-1 catalytic domain (SCD) via immobilized metal affinity chromatography under denaturing conditions that inhibit proteolytic activity followed by on-column refolding and spontaneous autolysis of the fusion peptide to yield pure, active stromelysin-1 catalytic domain. The methodology provides a general approach for the rapid purification of large quantities of zinc proteinases.     

The ketone cinnamoyl-(1-13C-Phe)-CGly-Pro-Pro is a tetrahedral transition state analog inhibitor of C. histolyticum collagenase

The ketone cinnamoyl-(1-13C-Phe)-CGly-Pro-Pro [(4-13C-5-cinnamido-4-oxo-6-phenylhexanoyl)-Pro-Pro 2] competitively inhibits a mixture of collagenases from Clostridium histolyticum with a Ki of 40 +/- 6 nM. 13C-nmr spectroscopy of the ketone in the presence of this collagenase shows a bound 13C resonance at 102.6 ppm and the resonance of the free ketone at 212 ppm. Ketone alone shows no trace (less than 0.5%) of a resonance in the region around 100 ppm. The bound resonance is displaceable by another competitive inhibitor. This ketone is thus a transition state analog which is rehybridized from trigonal planar to tetrahedral upon binding to collagenase.     

Identification of oxygen nucleophiles in tetrahedral intermediates: 2H and 18O induced isotope shifts in 13C NMR spectra of pepsin-bound peptide ketone pseudosubstrates

The synthetic ketone peptide analogue of pepstatin, isovaleryl-L-valyl-[3-13C]-(3-oxo-4S)-amino-6-methylheptanoyl-L-al anyl-isoamylamide is a strong inhibitor of aspartyl proteases. When the peptide is added to porcine pepsin in H2O at pH 5.1, the 13C NMR chemical shift of the ketone carbon moves from 208 ppm for the inhibitor in solution to 99.07 ppm when bound to the enzyme active site. In 2H2O the bound shift is 98.71 ppm, 0.36 ppm upfield. For the analogous experiment contrasting H216O and H218O, the 13C chemical shift was 0.05 ppm to higher field for the heavier isotope. These data show that water, and not an enzyme nucleophile, adds to the peptide carbonyl to yield a tetrahedral diol adduct in the enzyme-catalyzed reaction, and provide a method for differentiating between covalent and non-covalent mechanisms.     

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453 have been solved at 2.01 and 2.37A resolutions, respectively. The results revealed that the binding modes for this inhibitor to MMP-3 and -13 were quite similar. However, subtle comparative differences were observed at the bottom of S1' pockets, which were occupied with the guanidinomethyl moiety of the inhibitor. A remarkable feature of the inhibitor was the deep penetration of its long aliphatic chain into the S1' pocket and exposure of the guanidinomethyl moiety to the solvent.     

13C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein

13C NMR has been used to observe the equilibrium complex of [4-13C]-5-aminolevulinate ([4-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [4-13C]ALA (chemical shift = 205.9 ppm) forms [3,5-13C]PBG (chemical shifts = 121.0 and 123.0 ppm). PBG prepared from a mixture of [4-13C]ALA and [15N]ALA was used to assign the 121.0 and 123.0 ppm resonances to C5 and C3, respectively. For the enzyme-bound equilibrium complex formed from holoenzyme and [4-13C]ALA, two peaks of equal area with chemical shifts of 121.5 and 127.2 ppm are observed (line widths approximately 50 Hz), indicating that the predominant species is probably a distorted form of PBG. When excess free PBG is present, it is in slow exchange with bound PBG, indicating an exchange rate of less than 10 s-1, which is consistent with the turnover rate of the enzyme. For the complex formed from [4-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation, the predominant species is a Schiff base adduct (chemical shift = 166.5 ppm, line width approximately 50 Hz). Free ALA is in slow exchange with the Schiff base. Activation of the MMTS-modified enzyme-Schiff base complex with 113Cd and 2-mercaptoethanol results in the loss of the Schiff base signal and the appearance of bound PBG with the same chemical shifts as for the bound equilibrium complex with Zn(II) enzyme. Neither splitting nor broadening from 113Cd-13C coupling was observed.     

Nuclear magnetic resonance signal assignments of purified [13C]methyl-enriched yeast phenylalanine transfer ribonucleic acid

Yeast tRNA Phe, enriched in carbon-13 specifically at the naturally occurring methyl groups, has been produced through biosynthesis, then purified, and analyzed. Transfer RNA Phe was purified from the [13C]methyl-enriched, unfractionated tRNA that had been extracted from a methionine auxotroph of Saccharomyces cerevisiae [Agris, P. F., Kovacs, S. A. H., Smith, C., Kopper, R. H., & Schmidt, P. G. (1983) Biochemistry 22, 1402-1408]. The yeast had been grown in minimal medium supplemented with [13C]methylmethionine. Transfer RNA Phe purity and the full extent of nucleoside modification were confirmed by high-performance liquid chromatography of constituent nucleosides with simultaneous UV spectral identification and quantitation. Mass spectometry of [13C]methyl-enriched nucleosides and NMR of the tRNA indicated an enrichment of at least 70 atom %. Twelve resolved and prominent carbon-13 NMR signals from the tRNA were seen between 10 and 60 ppm. These have been assigned to 13 of the 14 naturally occurring methyl groups. However, the partially resolved signals assigned to the two 5-methylcytidines could not be assigned to their specific nucleoside positions of either 40 or 49 in the molecule. In addition, the partially resolved signals of the two methyl esters of wybutosine could not be distinguished. The methyl group found not to be enriched with 13C is bound to the ring carbon in the hypermodified nucleoside wybutosine (Y). A 13th enriched signal downfield (120.9 ppm) has been assigned to one of the two carbons added to guanosine to form the third ring in the biosynthesis of Y. The 13C enrichment of this ring carbon demonstrates its origin from the methionine methyl group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the binding of hydroxamic acid and carboxylic acid inhibitors to the stromelysin-1 (matrix metalloproteinase-3) catalytic domain by isothermal titration calorimetry.

Matrix metalloproteinases (MMPs) are implicated in diseases such as arthritis and cancer. Among these enzymes, stromelysin-1 can also activate the proenzymes of other MMPs, making it an attractive target for pharmaceutical design. Isothermal titration calorimetry (ITC) was used to analyze the binding of three inhibitors to the stromelysin catalytic domain (SCD). One inhibitor (Galardin) uses a hydroxamic acid group (pK(a) congruent with 8.7) to bind the active site zinc; the others (PD180557 and PD166793) use a carboxylic acid group (pK(a) congruent with 4.7). Binding affinity increased dramatically as the pH was decreased over the range 5.5-7.5. Experiments carried out at pH 6.7 in several different buffers revealed that approximately one and two protons are transferred to the enzyme-inhibitor complexes for the hydroxamic and carboxylic acid inhibitors, respectively. This suggests that both classes of inhibitors bind in the protonated state, and that one amino acid residue of the enzyme also becomes protonated upon binding. Similar experiments carried out with the H224N mutant gave strong evidence that this residue is histidine 224. DeltaG, DeltaH, DeltaS, and DeltaC(p) were determined for the three inhibitors at pH 6.7, and DeltaC(p) was used to obtain estimates of the solvational, translational, and conformational components of the entropy term. The results suggest that: (1) a polar group at the P1 position can contribute a large favorable enthalpy, (2) a hydrophobic group at P2' can contribute a favorable entropy of desolvation, and (3) P1' substituents of certain sizes may trigger an entropically unfavorable conformational change in the enzyme upon binding. These findings illustrate the value of complete thermodynamic profiles generated by ITC in discovering binding interactions that might go undetected when relying on binding affinities alone.     

13C NMR studies of methylene and methine carbons of substrate bound to a 280,000-dalton protein, porphobilinogen synthase

13C NMR has been used to observe the equilibrium complex of [5,5-2H,5-13C]-5-aminolevulinate [( 5,5-2H,5-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [5,5-2H,5-13C]ALA (chemical shift 46.9 ppm in D2O) was prepared from [5-13C]ALA through enolization in deuteriated neutral potassium phosphate buffer. In the PBG synthase reaction [5,5-2H,5-13C]ALA forms [2,11,11-2H,2,11-13C]PBG (chemical shifts 116.2 ppm for C2 and 34.2 ppm for C11 in D2O). For the complex formed between [5,5-2H,5-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation but can form a Schiff base adduct, the chemical shift of 44.2 ppm (line width 92 Hz) identifies an imine structure as the predominant tautomeric form of the Schiff base. By comparison to model compounds, the stereochemistry of the imine has been deduced; however, the protonation state of the imine nitrogen remains unresolved. Reconstitution of the MMTS-modified enzyme-Schiff base complex with Zn(II) and 2-mercaptoethanol results in the holoenzyme-bound equilibrium complex; this complex contains predominantly enzyme-bound PBG, and spectra reveal two peaks from bound PBG and two from free PBG. For bound PBG, C2 is -2.8 ppm from the free signal and C11 is +2.6 ppm from the free signal; the line widths of the bound signals are 55 and 75 Hz, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum

The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.     

Correlation of low-barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin

The structures of the hemiketal adducts of Ser 195 in chymotrypsin with N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone (AcLF-CF3) and N-acetyl-L-phenylalanyl trifluoromethyl ketone (AcF-CF3) were determined to 1.4-1.5 A by X-ray crystallography. The structures confirm those previously reported at 1.8-2.1 A [Brady, K., Wei, A., Ringe, D., and Abeles, R. H. (1990) Biochemistry 29, 7600-7607]. The 2.6 A spacings between Ndelta1 of His 57 and Odelta1 of Asp 102 are confirmed at 1.3 A resolution, consistent with the low-barrier hydrogen bonds (LBHBs) between His 57 and Asp 102 postulated on the basis of spectroscopy and deuterium isotope effects. The X-ray crystal structure of the hemiacetal adduct between Ser 195 of chymotrypsin and N-acetyl-L-leucyl-L-phenylalanal (AcLF-CHO) has also been determined at pH 7.0. The structure is similar to the AcLF-CF3 adduct, except for the presence of two epimeric adducts in the R- and S-configurations at the hemiacetal carbons. In the (R)-hemiacetal, oxygen is hydrogen bonded to His 57, not the oxyanion site. On the basis of the downfield 1H NMR spectrum in solution, His 57 is not protonated at Nepsilon2, and there is no LBHB at pH >7.0. Because addition of AcLF-CHO to chymotrypsin neither releases nor takes up a proton from solution, it is concluded that the hemiacetal oxygen of the chymotrypsin-AcLF-CHO complex is a hydroxyl group and not attracted to the oxyanion site. The protonation states of the hemiacetal and His 57 are explained by the high basicity of the hemiacetal oxygen (pK(a) > 13.5) relative to that of His 57. The 13C NMR signal for the adduct of AcLF-13CHO with chymotrypsin is consistent with a neutral hemiacetal between pH 7 and 13. At pH <7.0, His 57 in the AcLF-CHO-hemiacetal complex of chymotrypsin undergoes protonation at Nepsilon2 of His 57, leading to a transition of the 15.1 ppm downfield signal to 17.8 ppm. The pK(a)s in the active sites of the AcLF-CF3 and AcLF-CHO adducts suggest an energy barrier of 6-7 kcal x mol(-1) against ionizations that change the electrostatic charge at the active site. However, ionizations of neutral His 57 in the AcLF-CHO-chymotrypsin adduct, or in free chymotrypsin, proceed with no apparent barrier. Protonation of His 57 is accompanied by LBHB formation, suggesting that stabilization by the LBHB overcomes the barrier to ionization. On the basis of the hydration constant for AcLF-13CHO and its inhibition constant, its K(d) is 16 microM, 8000-fold larger than the comparable value for AcLF-CF3.     

Structural changes that occur upon photolysis of the Fe(II)(a3)-CO complex in the cytochrome ba(3)-oxidase of Thermus thermophilus: a combined X-ray crystallographic and infrared spectral study demonstrates CO binding to Cu(B)

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a(3) moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba(3)-oxidase from Thermus thermophilus, determined at ~2.8-3.2? resolution, reveal a Fe-C distance of ~2.0?, a Cu-O distance of 2.4? and a Fe-C-O angle of ~126°. Upon photodissociation at 100K, X-ray structures indicate loss of Fe(a3)-CO and appearance of Cu(B)-CO having a Cu-C distance of ~1.9? and an O-Fe distance of ~2.3?. Absolute FTIR spectra recorded from single crystals of reduced ba(3)-CO that had not been exposed to X-ray radiation, showed several peaks around 1975cm(-1); after photolysis at 100K, the absolute FTIR spectra also showed a significant peak at 2050cm(-1). Analysis of the 'light' minus 'dark' difference spectra showed four very sharp CO stretching bands at 1970cm(-1), 1977cm(-1), 1981cm(-1), and 1985cm(-1), previously assigned to the Fe(a3)-CO complex, and a significantly broader CO stretching band centered at ~2050cm(-1), previously assigned to the CO stretching frequency of Cu(B) bound CO. As expected for light propagating along the tetragonal axis of the P4(3)2(1)2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba(3) crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO(2) at 2337cm(-1) and one from traces of CO at 2133cm(-1); while bands associated with CO bound to either Fe(a3) or to Cu(B) in "light" minus "dark" FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2? and FTIR spectra support the long-held position that photolysis of Fe(a3)-CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu(B) atom; Fe(a3) and Cu(B) ligation, at the resolutions reported here, are otherwise unaltered.     

Synthesis of a new 8-spin-labeled analog of adenosine 5'-phosphate and its interaction with AMP nucleosidase.

The synthesis of a new 8-spin-labeled analog of AMP, 8-[[[(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)carbamoyl]methyl]thio]adenosine 5'-phosphate (8-slAMP), is described. The procedure is facile and results in high yields. 8-slAMP is a competitive inhibitor of AMP nucleosidase with a Ki of 19 microM as compared to a Km of 100 microM for AMP. The analog is not a substrate for the enzyme and does not displace MgATP2- from the allosteric sites under the usual assay conditions. The EPR spectrum of the bound spin probe reveals a highly immobilized nitroxide group. Binding studies with 8-slAMP at 8 degrees C indicate three independent binding sites (Kd = 1.4 microM) per molecule of enzyme (Mr = 320,000). These properties make 8-slAMP a good spin probe for AMP nucleosidase. The analog may also be useful for other proteins known or suspected of binding AMP analogs in a syn conformation.