Inhibition of collagenase from Clostridium histolyticum by phosphoric and phosphonic amides

Di- and tripeptides with sequences present in collagen that are known to occupy the S1' through S3' subsites at the active site of the collagenase from Clostridium histolyticum do not themselves inhibit this zinc protease. Thus glycylproline, glycylprolylalanine, and their C-terminal amides are not inhibitors. N alpha-Phosphorylglycylproline, N alpha-phosphorylglycyl-L-prolyl-L-alanine, and their C-terminal amides are weak inhibitors with IC50's (concentration causing half-maximal inhibition) of 4.6, 0.8, 3, and 1.5 mM, respectively. Extension of glycyl-L-prolyl-L-alanine to L-leucyl-glycyl-L-prolyl-L-alanine gives a tetrapeptide known to occupy the S1, S1', S2', and S3' subsites of collagenase when present in collagen but that still does not itself inhibit the enzyme. (Isoamylphosphonyl)glycyl-L-prolyl-L-alanine, a peptide containing a tetrahedral phosphorus atom at the position of the amide carbonyl carbon of the L-leucylglycyl amide bond of the parent tetrapeptide, inhibits collagenase with an IC50 of 16 microM, at least 1000-fold more potent than the parent peptide. Substitution of the two-carbon ethyl chain of alanine for the five-carbon isoamyl chain of leucine increases the IC50 to 46 microM. Substitution of the n-decyl chain for the isoamyl chain does not change the IC50. (Isoamylphosphonyl)glycyl-glycyl-L-proline contains a tripeptide that does not occupy the S1' through S3' subsites of collagenase when this peptide is present in collagen and thus has an IC50 of 4.4 mM. (Isoamylphosphonyl)glycyl-L-prolyl-L-alanine may be an analogue of the tetrahedral transition state for the hydrolysis of the natural collagen substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aldehyde and ketone substrate analogues inhibit the collagenase of Clostridium histolyticum

The collagenase from Clostridium histolyticum is a mixture of several collagenases, all of which are zinc metalloproteases. This enzyme catalyzes the cleavage of the X-Gly peptide bond in the repeating sequence of collagen: -Gly-Pro-X-Gly-Pro-X-. Thus the S3, S2, and S1 subsites on the enzyme appear to be occupied by the sequence -Gly-Pro-X- and the S1', S2', and S3' subsites also by -Gly-Pro-X-. Short peptides up to and including N alpha-acyltetrapeptides containing the repeat sequence do not detectably inhibit the enzyme (IC50 greater than 10 mM). However, peptide aldehydes of the form aminoacyl-X-glycinal, presumably occupying the S1, S2, ..., Sn subsites, are inhibitors. The most potent of these was Pro6-Gly-Pro-glycinal, with an IC50 of 340 +/- 70 microM. The single peptide aldehyde investigated, which could occupy the S1' and S2' subsites, 4-oxobutanoyl-L-proline, did not inhibit collagenase (IC50 greater than 20 mM). The peptide ketone 5-benzamido-4-oxo-6-phenylhexanoyl-Pro-Ala (XXV), which could occupy the S1-S3' subsites, inhibits collagenase with an IC50 of 120 +/- 50 microM, over 80-fold more potently than its parent peptide analogue benzoyl-Phe-Gly-Pro-Ala (XXIII). The alcohol analogue of XXV, 5-benzamido-4-hydroxy-6-phenylhexanoyl-Pro-Ala (XXVI), is over 60-fold less potent with an IC50 of 8 +/- 2mM. Extending the peptide ketone XXV to occupy the S2-S3' subsites gave 5-(N alpha-carbobenzoxy-L-prolinamido)-4-oxo-6-phenylhexanoyl-Pro -Ala (XXVII). Surprisingly, XXVII had an IC50 of only 5.2 +/- 2 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of angiotensin converting enzyme by phosphoramidates and polyphosphates

N alpha-Phosphoryl-L-alanyl-L-proline is a reversible competitive inhibitor of angiotensin converting enzyme with a Ki of 1.4 nM. Alkylation of one phosphate oxygen with methyl, ethyl, or benzyl does not change the Ki. The high activity of the O-alkylated inhibitors demonstrates that the two phosphate oxygen anions do not constitute a bidentate ligand of the active site zinc ion. Substitution of valyltryptophan, glycylglycine, or delta-aminovaleric acid for alanylproline in the phosphoramidate raises the Ki to 12 nM, 25 microM, and 178 microM, respectively. Methylation of the alanine nitrogen in phosphorylalanylproline raises the Ki to 29 microM. Polyphosphates inhibit converting enzyme with the following Ki's: phosphate, approximately 300 mM; pyrophosphate, 2 mM; tripolyphosphate, 18 microM; tetrapolyphosphate, 150 microM. The inhibition by tripolyphosphate appears to be competitive and is unaffected by the addition of excess zinc ion. Since the Ki of tripolyphosphate is nearly 10-fold lower than that of N-phosphoryl-delta-aminovaleric acid and is near that of N alpha-phosphorylglycylglycine, its terminal phosphates may bind the zinc site and the cationic site on the enzyme, thus spanning the S1' and S2' sites.     

alpha-Bromo ketone substrate analogues are powerful reversible inhibitors of carboxypeptidase A

The aldehyde (RS)-2-benzyl-4-oxobutanoic acid, which is 25% hydrated at pH 7.5, has recently been shown to be a strong reversible competitive inhibitor of carboxypeptidase A [Ki = 0.48 nM; Galardy, R. E., & Kortylewicz, Z. P. (1984) Biochemistry 23, 2083-2087]. The ketone analogue of this aldehyde (RS)-2-benzyl-4-oxopentanoic acid (IV) is not detectably hydrated under the same conditions and is 1500-fold less potent (Ki = 730 microM). The ketone homologue (RS)-2-benzyl-5-oxohexanoic acid (XIII) is also a weak inhibitor (Ki = 1.3 mM). The alpha-monobrominated derivatives of these two ketones are, however, strong competitive inhibitors with Ki's of 0.57 microM and 1.3 microM, respectively. Oximes derived from the aldehyde, the ketones IV and XIII, and a homologue of the aldehyde are weak inhibitors with Ki's ranging from 480 to 7900 microM. The inhibition of carboxypeptidase A by the alpha-monobrominated ketones is reversible and independent of the time (up to 6 h) of incubation of enzyme and inhibitor together. Bromoacetone at a concentration of 30 mM does not inhibit carboxypeptidase A. Incubation of an equimolar mixture of 2-benzyl-4-bromo-5-oxohexanoic acid (XV) and enzyme for 1 h led to the recovery of 82% of XV, demonstrating that it is the major species reversibly bound during assay of inhibition. Taken together, these results indicate that tight binding of carbonyl inhibitors to carboxypeptidase A requires specific binding of inhibitor functional groups such as benzyl and an electrophilic carbonyl carbon such as that of an alpha-bromo ketone or aliphatic aldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extended binding inhibitors of chymotrypsin that interact with leaving group subsites S1'-S3'

We have synthesized inhibitors of chymotrypsin, based on fluoromethyl ketones, that bind at S and S' subsites. "Small" inhibitors of serine proteases, which have previously been synthesized, only interact with S subsites. The parent compound is Ac-Leu-ambo-Phe-CF2H (1) (Ki = 25 X 10(-6) M). This inhibitor was modified by successively replacing H of the -CF2H group by -CH2CH2CONHCH3, (4), -CH2CH2CONH-Leu-NHMe (5), -CH2CH2CONH-Leu-Val-OEt (6), and -CH2CH2CONH-Leu-Arg-OMe (7). Corresponding Ki values are 7.8 (4), 0.23 (5), 0.21 (6), and 0.014 (7) microM. Extending 5 to 6 by addition of Val-OEt at P3' does not decrease Ki. In contrast, extension of 5 to 7 by incorporating Arg-OMe at P3' decreases Ki approximately 15-fold, suggesting interaction between Arg and the S3' subsite but no corresponding interaction at that subsite with Val. These results are in accordance with results obtained with the homologous family of avian ovomucoid third domain proteins. Proteins with Arg at the P3' position show highly favorable interactions with the protease at the S3' subsite [Park, S. J. (1985) Ph.D. Thesis, Purdue University; M. Laskowski, Jr., personal communication]. These results establish that incorporation of residues which interact with S' subsites significantly increases the efficacy of inhibitors and that valuable information concerning the most effective amino acid composition of small inhibitors can be obtained from the amino acid sequence of protein inhibitors.     

Active site directed N-carboxymethyl peptide inhibitors of a soluble metalloendopeptidase from rat brain

A soluble metalloendopeptidase isolated from rat brain preferentially cleaves bonds in peptides having aromatic residues in the P1 and P2 position. An additional aromatic residue in the P3' position greatly increases the binding affinity of the substrate, suggesting the presence of an extended substrate recognition site in the enzyme, capable of binding a minimum of five amino acid residues [Orlowski, M., Michaud, C., & Chu, T.G. (1983) Eur. J. Biochem. 135, 81-88]. A series of N-carboxymethyl peptide derivatives structurally related to model substrates and containing a carboxylate group capable of coordinating with the active site zinc atom were synthesized and tested as potential inhibitors. One of these inhibitors, N-[1(RS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, was found to be a potent competitive inhibitor of the enzyme with a Ki of 1.94 microM. The two diastereomers of this inhibitor were separated by high-pressure liquid chromatography. The more potent diastereomer had a Ki of 0.81 microM. The inhibitory potency of the less active diastereomer was lower by 1 order of magnitude. Decreasing the hydrophobicity of the residue binding the S1 subsite of the enzyme by, for example, replacement of the phenylethyl group with a methyl residue decreased the inhibitory potency by almost 2 orders of magnitude. Deletion of the carboxylate group decreased the inhibitory potency by more than 3 orders of magnitude. Shortening the inhibitor chain by a single alanine residue had a similar effect. Binding of the inhibitor to the enzyme increased its thermal stability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of arginine aminopeptidase by bestatin and arphamenine analogues. Evidence for a new mode of binding to aminopeptidases

The synthesis and inhibition kinetics of a new, potent inhibitor of arginine aminopeptidase (aminopeptidase B; EC 3.4.11.6) are reported. The inhibitor is a reduced isostere of bestatin in which the amide carbonyl is replaced by the methylene (-CH2-) moiety. Analysis of the inhibition of arginine aminopeptidase by this inhibitor according to the method of Lineweaver and Burk yields an unusual noncompetitive double-reciprocal plot. The replot of the slopes versus [inhibitor] is linear (Kis = 66 nM), but the replot of the y intercepts (1/V) versus [inhibitor] is hyperbolic (Kii = 10 nM, Kid = 17 nM). These results provide evidence for a kinetic mechanism in which the inhibitor binds to the S1' and S2' subsites on the enzyme, not the S1 and S1' subsites occupied by dipeptide substrates. Furthermore, structure-activity data for a series of ketomethylene dipeptide isosteres in which the amide (-CONH-) of a dipeptide is replaced with the ketomethylene (-COCH2-) moiety show that the S1 and S1' subsites preferentially bind basic and aromatic side chains, respectively. These results are in agreement with the known substrate specificity of arginine aminopeptidase. The structure-activity data for several bestatin analogues, however, show that these compounds do not bind to the S1 and S1' sites of arginine aminopeptidase. A comparison of the data provides evidence that bestatin inhibits arginine aminopeptidase and possibly other aminopeptidases by binding to the S1' and S2' sites of the enzyme.     

Substrate/inhibitor properties of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2) towards the sugar moiety of nucleosides, including O'-alkyl analogues.

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono- and di-O'-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower Ki values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3'-hexanoylamino-2',3'-dideoxythymidine, with a Ki of approximately 600 microM for TK1 and approximately 0.1 microM for TK2. 3'-OMe-dC was a superior inhibitor of dCK to its 5'-O-methyl congener, consistent with possible participation of the oxygen of the (3')-OH or (3')-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly alpha-dT was a good substrate of both TK1 and TK2, with Ki values of 120 and 30 microM for TK1 and TK2, respectively; and a 3'-branched alpha-L-deoxycytidine analogue proved to be as good a substrate as its alpha-D-counterpart. Several 5'-substituted analogues of dC were good non-substrate inhibitors of dCK and, to a lesser extent, of TK2. Finally, some ribonucleosides are substrates of the foregoing enzymes; in particular C is a good substrate of dCK, and 2'-OMe-C is an even better substrate than dC.     

Slow- and fast-binding inhibitors of thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues

The modes of binding to thermolysin of two phosphonamidate peptide inhibitors, carbobenzoxy-GlyP-L-Leu-L-Leu (ZGPLL) and carbobenzoxy-L-PheP-L-Leu-L-Ala (ZFPLA), have been determined by X-ray crystallography and refined at high resolution to crystallographic R-values of 17.7% and 17.0%, respectively. (GlyP is used to indicate that the trigonal carbon of the peptide linkage is replaced by the tetrahedral phosphorus of a phosphonamidate group.). These inhibitors were designed to be structural analogues of the presumed catalytic transition state and are potent inhibitors of thermolysin (ZGPLL, Ki = 9.1 nM; ZFPLA, Ki = 0.068 nM) [Bartlett, P. A., & Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. ZFPLA binds to thermolysin in the manner expected for the transition state and, for the first time, provides direct support for the presumed mode of binding of extended substrates in the S2 subsite. The mode of binding of ZFPLA displays all the interactions that are presumed to stabilize the transition state and supports the postulated mechanism of catalysis [Hangauer, D. G., Monzingo, A. F., & Matthews, B. W. (1984) Biochemistry 23, 5730-5741]. The two oxygens of the phosphonamidate moiety are liganded to the zinc to give overall pentacoordination of the metal. For the second inhibitor the situation is different. Although both ZFPLA and ZGPLL have similar modes of binding in the S1' and S2' subsites, the configurations of the carbobenzoxy-Phe and carbobenzoxy-Gly moieties are different. For ZFPLA the carbonyl group of the carbobenzoxy group is hydrogen bonded directly to the enzyme, whereas in ZGPLL the carbonyl group is rotated 117 degrees, and there is a water molecule interposed between the inhibitor and the enzyme. For ZGPLL only one of the phosphonamidate oxygens is liganded to the zinc. Correlated with the change in inhibitor-zinc ligation from monodentate in ZGPLL to bidentate in ZFPLA there is an increase in the phosphorus-nitrogen bond length of about 0.25 A, strongly suggesting that the phosphonamide nitrogen in ZFPLA is cationic, analogous to the doubly protonated nitrogen of the transition state. The observation that the nitrogen of ZFPLA appears to donate two hydrogen bonds to the protein also indicates that it is cationic. The different configurations adopted by the respective inhibitors are correlated with large differences in their kinetics of binding [Bartlett, P. A., & Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. These differences in kinetics are not associated with any significant conformational change on the part of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of angiotensin converting enzyme by L-alanyl-4 or 5-substituted-L-prolines and their N alpha-phosphoryl-derivatives

A series of L-alanyl-4 or 5-substituted L-prolines, such as L-alanyl-L-thiazolidine-4-carboxylic acid, L-alanyl-5-oxo-L-proline, L-alanyl-trans-4-hydroxy-L-proline and L-alanyl-cis-4-hydroxy-L-proline as well as their corresponding N alpha-phosphoryl derivatives, were synthesized and studied for inhibition against angiotensin converting enzyme. Furanacryloyl-phenylalanyl-glycyl-glycine was used as substrate in 50 mM Tris hydrochloride buffer at pH 7.5 containing 1 microM zinc acetate. N alpha-Phosphoryl-L-alanyl-L-thiazolidine-4-carboxylic acid and N alpha-phosphoryl-L-alanyl-trans-4-hydroxy-L-proline competitively inhibit angiotensin converting enzyme with Ki values of 68 microM and 89.3 microM, respectively. Smaller inhibition against angiotensin converting enzyme was obtained with the rest of compounds studied here.     

Inhibitors of angiotensin-converting enzyme containing a tetrahedral arsenic atom.

A series of tetrahedral oxo acids of Group VA and VIA elements and of silicon and boron were examined as inhibitors of angiotensin-converting enzyme. Arsenate is a competitive inhibitor with a Ki of 27 +/- 1 mM, at least 10-fold more potent than phosphate. Dimethylarsinate is a competitive inhibitor with a Ki of 70 +/- 9 mM, 2-fold more potent than dimethylphosphinate. Oxo acids of boron, silicon, antimony, sulphur and selenium are not inhibitors. On the basis of these results and the strong inhibition of this zinc metallopeptidase by substrate analogues containing a tetrahedral phosphorus atom, two substrate analogues containing a tetrahedral arsenic atom were prepared. 2-Arsonoacetyl-L-proline is a competitive inhibitor with a Ki of 18 +/- 7 mM, more than 2000-fold weaker than that of its phosphorus analogue 2-phosphonoacetyl-L-proline. 4-Arsono-2-benzylbutanoic acid is a mixed inhibitor with a Ki of 0.5 +/- 0.2 mM, indistinguishable in potency from its phosphorus analogue 2-benzyl-4-phosphonobutanoic acid.     

Fluoro ketone inhibitors of hydrolytic enzymes

The use of fluoro ketones as inhibitors of hydrolytic enzymes has been investigated. The acetylcholine analogues 6,6-dimethyl-1,1,1-trifluoro-2-heptanone and 3,3-difluoro-6,6-dimethyl-2-heptanone are inhibitors of acetylcholinesterase with Ki values of 16 X 10(-9) M and 1.6 X 10(-9) M, respectively. These fluoro ketones are 10(4)-10(5) times better as inhibitors than the corresponding methyl ketone. Since nucleophiles readily add to fluoro ketones, it is likely that these compounds inhibit acetylcholinesterase by formation of a stable hemiketal with the active-site serine residue. Fluoro ketone substrate analogues are also inhibitors of zinc metallo- and aspartylproteases. 2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is an inhibitor of carboxypeptidase A (Ki = 2 X 10(-7) M). Trifluoromethyl ketone dipeptide analogues are good inhibitors of angiotensin converting enzyme. An analogue of pepstatin that contains a difluorostatone residue in place of statine has been prepared and found to be an extremely potent inhibitor of pepsin (Ki = 6 X 10(-11) M). The hydrated ketones are probably the inhibitory species since they are structural mimics of the tetrahedral intermediate that forms during the hydrolysis of peptide substrates.     

Characterization of a membrane-bound aminopeptidase purified from Acyrthosiphon pisum midgut cells. A major binding site for toxic mannose lectins

A single membrane-bound aminopeptidase N (APN) occurs in the pea aphid (Acyrthosiphon pisum Harris) midgut, with a pH optimum of 7.0, pI of 8.1 and molecular mass of 130 kDa. This enzyme accounts for more than 15.6% of the total gut proteins. After being solubilized in detergent, APN was purified to homogeneity. The enzyme is a glycoprotein rich in mannose residues, which binds the entomotoxic lectins of the concanavalin family. The internal sequence of APN is homologous with a conservative domain in APNs, and degenerated primers of highly conserved APN motifs were used to screen a gut cDNA library. The complete sequence of APN has standard residues involved in zinc co-ordination and catalysis and a glycosyl-phosphatidylinositol anchor, as in APNs from Lepidoptera. APN has a broad specificity towards N-terminal amino acids, but does not hydrolyze acidic aminoacyl-peptides, thus resembling the mammalian enzyme (EC 3.4.11.2). The kcat/Km ratios for different di-, tri-, tetra-, and penta-peptides suggest a preference for tripeptides, and that subsites S1, S2' and S3' are pockets able to bind bulky aminoacyl residues. Bestatin and amastatin bound APN in a rapidly reversible mode, with Ki values of 1.8 microM and 0.6 microM, respectively. EDTA inactivates this APN (k(obs) 0.14 M(-1) x s(-1), reaction order of 0.44) at a rate that is reduced by competitive inhibitors. In addition to oligopeptide digestion, APN is proposed to be associated with amino-acid-absorption processes which, in contrast with aminopeptidase activity, may be hampered on lectin binding.     

Dehydroquinate synthase: the use of substrate analogues to probe the late steps of the catalyzed reaction

The later steps of the proposed mechanistic pathway for the reaction catalyzed by dehydroquinate synthase have been probed by using three substrate analogues. Each of these analogues is structurally prohibited from undergoing the ring-opening reaction that necessarily precedes the carbon-carbon bond-forming step in the overall conversion of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) to dehydroquinate (2). Two of the analogues (the 2-deoxy cyclic compound 3 and the carbacyclic material 4) are locked into a cyclic form, mimicking the pyranose form of the substrate DAHP. The third analogue, 5, contains no carbonyl group at C-2 and may thus resemble the open-chain form of DAHP. Analogues 3 and 4 each bind to the enzyme and are competitive inhibitors having Ki values of 35 and 0.12 microM, respectively. More importantly, however, incubation of these analogues with the enzyme leads to the catalytic production of Pi along with the corresponding exomethylene compounds that are analogous to the enol ether IV postulated for the normal synthase reaction. In contrast to these results, the acyclic analogue 5 is neither a substrate nor an inhibitor of the enzyme. These data suggest that the enzyme recognizes and acts upon the alpha-pyranose form of the natural substrate. The ready release of the exomethylene products from the processing of analogues 3 and 4 is consistent with the suggestion of Bartlett and his group that the enzyme may release the enol ether intermediate IV into solution, where the ring opening and cyclization occur nonenzymically. The use of 3 stereospecifically labeled with deuterium at C-7 allows the sterochemical course of the beta-elimination of phosphate to be established. This step proceeds with syn stereochemistry, which fits the pattern of enzyme-catalyzed elimination from substrates where the proton is lost from a position alpha to a ketone, an aldehyde, or a thiolester. Since the overall stereochemical course of the transformation mediated by dehydroquinate synthase had been shown to be inversion, the present finding of a syn elimination suggests that the transition state for the subsequent intramolecular aldol reaction has a chairlike geometry.     

Inhibition of cGMP-dependent protein kinase by (Rp)-guanosine 3',5'-monophosphorothioates

The activation of the cGMP-dependent protein kinase and cAMP-dependent protein kinase by the diastereomers of guanosine 3',5'-monophosphorothioate, (Sp)-cGMPS and (Rp)-cGMPS, and 8-chloroguanosine 3',5'-monophosphorothioate, (Sp)-8-Cl-cGMPS and (Rp)-8-Cl-cGMPS, was investigated using the peptide Kemptide as substrate. The (Sp)-diastereomers, which have an axial exocyclic sulfur atom, bound to the cGMP-dependent protein kinase and stimulated its phosphotransferase activity. In contrast, the (Rp)-isomers, which have an equatorial exocyclic sulfur atom, bound to the enzyme without stimulation of its activity. (Rp)-cGMPS and (Rp)-8-Cl-cGMPS antagonized the activation of the cGMP-dependent protein kinase with a Ki of 20 microM and 1.5 microM, respectively. (Rp)-cGMPS also antagonized the activation of cAMP-dependent protein kinase with a Ki of 20 microM. In contrast, (Rp)-8-cGMPS ws a weak inhibitor of the cAMP-dependent protein kinase with a Ki of 100 microM. (Rp)-8-Cl-cGMPS appears to be a rather selective inhibitor of the cGMP-dependent protein kinase and may be a useful tool for studying the role of cGMP in broken and intact cell systems.     

Substrate and inhibitor specificity of 3-hydroxy-3-methylglutaryl-CoA reductase determined with substrate-analogue CoA-thioesters and CoA-thioethers.

1) Analogues of 3-hydroxy-3-methylglutaryl-CoA were prepared in which the substituents at C-3 of the acyl residue were altered. The same analogues were additionally modified by replacement of the thioester oxygen by hydrogen to yield reduction-resistant CoA-thioethers. The interaction of both types of CoA derivatives with a 58-kDa catalytic fragment of human 3-hydroxy-3-methylglutaryl-CoA reductase was studied. 2) This enzyme reduces glutaryl-CoA at a very low rate whereas 3-hydroxyglutaryl-CoA is well reduced, the maximal rate of reduction being 7% that of the physiological substrate. Only half of total 3-hydroxyglutaryl-CoA was attacked, thus reflecting the stereo-specificity of the enzyme for (3S)-3-hydroxy-3-methylglutaryl-CoA. The results invalidate the hitherto assumed absolute substrate specificity of the enzyme. 3) The affinity of both 3-hydroxyglutaryl-CoA and its thioether variant S-(4-carboxy-3-hydroxybutyl)CoA to the reductase, Ki = 0.3 microM and Ki = 0.4 microM, respectively, is higher than that of the physiological substrate, Km = 1.5 microM (data related to (S)-diastereomer). The results show for the first time that the methyl-group effect observed with the inhibitor lovastatin is an intrinsic property of the enzyme. 4) All of the prepared CoA derivatives are purely competitive inhibitors of the reductase, the affinities varying within a range of two powers of ten (Ki = 0.3-32 microM). On variation of the substituents at C-3 of the acyl residue of the physiological substrate the affinity of both CoA-thioesters and CoA-thioethers increases in the sequence CH2, C(CH3)2, CH(CH3), C(OH)CH3, CH(OH).     

Enzymatic synthesis and inhibitory characteristics of tartronate semialdehyde phosphate

The immediate product of the pyruvate kinase catalyzed phosphorylation of beta-hydroxypyruvate is the enol of tartronate semialdehyde phosphate (TSP). The reaction has the same pH profile as that for the phosphorylation of pyruvate with pK's of 8.2 and 9.7 observed in H2O. This enol tautomerizes in solution to the aldehyde, which in turn becomes hydrated. 31P NMR spectra indicate that the enol resonates approximately 1 ppm upfield from the hydrated aldehyde. By following the tautomerization spectrophotometrically at 240 nm, we have found it to be independent of pH (0.2 min-1 below pH 6 in water), except that it is 2-fold slower above the pK of the phosphate group (6.3 in H2O and 6.7 in D2O). It is 3.6-fold slower in D2O. When this TSP is reduced with NaBH4, approximately 50% of the product is D-2-phosphoglyceric acid (substrate for enolase). Thus, while the immediate product of the phosphorylation rection is the enol of TSP, the eventual product is D,L-TSP. Both the enol and the aldehyde forms of TSP were found to be potent inhibitors of yeast enolase with apparent Ki values of 100 nM and 5 microM, respectively. However, since the aldehyde form is 95-99% hydrated [Stubbe, J., & Abeles, R. (1980) Biochemistry 19, 5505], the true Ki for the aldehyde species is 50-250 nM. The enol of TSP shows slow binding behavior, as expected for an intermediate analogue, with a t1/2 for this process of approximately 15 s (k = 0.046 s-1) and an initial Ki of approximately 200 nM.     

The roles of active site hydrogen bonding in cytochrome P-450cam as revealed by site-directed mutagenesis

The role of the active site hydrogen bond of cytochrome P-450cam has been studied utilizing a combination of site-directed mutagenesis and substrate analogues with altered hydrogen bonding capabilities. Cytochrome P-450cam normally catalyzes the regiospecific hydroxylation of the monoterpene camphor. The x-ray crystal structure of this soluble bacterial cytochrome P-450 (Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260, 16122-16128) indicates a specific hydrogen bond between tyrosine 96 and the carbonyl moiety of the camphor substrate. The site-directed mutant in which tyrosine 96 has been changed to a phenylalanine and the substrate analogues thiocamphor and camphane have been used to probe this interaction in several aspects of catalysis. At room temperature, both the mutant enzyme with camphor and the wild type enzyme with thiocamphor bound result in 59 and 65% high-spin ferric enzyme as compared to the 95% high spin population obtained with native enzyme and camphor as substrate. The equilibrium dissociation constant is moderately increased, from 1.6 microM for the wild type protein to 3.0 and 3.3 microM for wild type-thiocamphor and mutant-camphor complexes, respectively. Camphane bound to cytochrome P-450cam exhibits a larger decrease in high spin fraction (45%) and a correspondingly larger KD (46 microM), suggesting that the carbonyl moiety of camphor plays an important steric role in addition to its interaction as a hydrogen bond acceptor. The absolute regioselectivity of the mutant enzyme, and of the wild type enzyme with thiocamphor, is lost resulting in production of several hydroxylated products in addition to the 5-exo-hydroxy isomer. Based on rates of NADH oxidation, comparison of the substrate specificity for these systems (kcat/KD) indicates a 5- and 7-fold decrease in specificity for the mutant enzyme and thiocamphor-wild type complex, respectively. The replacement of the cytochrome P-450cam active site tyrosine with phenylalanine does not affect the branching ratio of monooxygenase versus oxidase chemistry or peroxygenase activity (Atkins, W.M., and Sligar, S.G. (1987) J. Am. Chem. Soc. 109, 3754-3760).     

Transition-state analogues as inhibitors of L-dopa decarboxylase

1. A series of compounds has been prepared which are analogues of the transition state of the reaction catalysed by L-dopa decarboxylase (EC 4.1.1.28). 2. These compounds are reduced adducts of the substrate (L-dopa) and coenzyme (pyridoxal phosphate), as well as analogues of these substances (D-dopa, pyridoxal and salicaldehyde). 3. Compounds were also prepared with an oxazine link between the 3'-oxygen and the nitrogen attached to the 4'-carbon of the aldehyde moiety. 4. None of the D-dopa adducts produced any significant inhibition, but the L-dopa adducts were all active at millimolar levels, with the oxazine derivatives being more active than their parent compounds. 5. Inhibition was competitive with respect to L-dopa, but was neither competitive nor non-competitive with respect to pyridoxal phosphate. 6. The most active compound tested was the oxazine derivative of the L-dopa/salicaldehyde adduct, with an estimated Ki of 58.0 microM. 7. Increased inhibitory activity was observed when enzyme depleted of pyridoxal phosphate was used.     

Comparison of inhibitory effects of prolinal-containing peptide derivatives on prolyl endopeptidases from bovine brain and Flavobacterium

The inhibitory effects of proline-containing peptides and their derivatives on prolyl endopeptidases from Flavobacterium meningosepticum and bovine brain were compared. Replacement of the carboxyl terminal proline in N-blocked peptides with prolinal resulted in remarkable decreases in Ki values for both prolyl endopeptidases. Further reduction of the prolinal to prolinol led to a decrease in their inhibitory effects. Z-Pro-, Z-Val-, and Suc-Pro-prolinals were similarly inhibitory for both the enzymes with Ki values of nM order. However, the inhibitory effects of Z-Pyr-prolinal and Boc-Pro-prolinal on these enzymes were significantly distinguished: they strongly inhibited the mammalian prolyl endopeptidase with Ki values of nM order, while the Ki values of these compounds for the microbial enzyme were only of microM order. These results suggest that there are some structural differences in the S2 and S3 subsites between the two enzymes, though their substrate specificities are apparently indistinguishable.