Biomedical subjects

D S Lawrence

Publications and source records attributed to D S Lawrence.

At least 55 records · Page 3Linked to original sources

PubMed1995

The extraordinary active site substrate specificity of pp60c-src. A multiple specificity protein kinase.

We report the first active site substrate specificity analysis of a tyrosine-specific protein kinase, namely pp60c-src. Like the cAMP-dependent protein kinase and protein kinase C, pp60c-src will phosphorylate an assortment of achiral residues attached to active site-directed peptides. Furthermore, pp60c-src phosphorylates both aromatic and aliphatic alcohols. However, the substrate specificity of pp60c-src is much broader than that of the two previously examined serine/threonine-specific protein kinases. We have previously shown that both the cAMP-dependent protein kinase and protein kinase C will utilize a wide array of non-amino acid residues as substrates, as long as the distance between the hydroxyl moiety and the adjacent peptide backbone is comparable with that present in serine and threonine (Kwon, Y.-G., Mendelow, M., and Lawrence, D. S. (1994) J. Biol. Chem. 269, 4839-4844). In marked contrast, pp60c-src does not discriminate against substrates on the basis of chain length, catalyzing the phosphorylation of residues that contain anywhere from 2-12 carbons between the alcohol functional group and the adjacent peptide bond. In addition, pp60c-src phosphorylates L-serine in an active site-directed peptide. The possible structural basis for the multiple specificity of pp60c-src is discussed. Finally, the active site specificity of pp60c-src is not just limited to L-amino acid residues, but also extends into the realm of D-amino acids as well.

Alcohols↗

PubMed1995

Photosensitization and tissue distribution studies of the picket fence porphyrin, 3,1-TPro, a candidate for photodynamic therapy.

From a structurally distinct set of o-substituted tetraphenylporphyrins, the picket fence porphyrin (PFP), 3,1-meso-tetrakis(o-propionamidophenyl)porphyrin (3,1-TPro) has been selected as a potential candidate for use in the photodynamic therapy (PDT) of cancer. In this report, the time-dependent tissue distribution of 14C-labeled 3,1-TPro is described along with the results of various treatment regimens. The tissue distribution of radiolabeled 3,1-TPro is comparable to that of other porphyrin photosensitizers with the advantage of being most effective at 4 h and being cleared rapidly from most tissues. The results of the various treatment regimen experiments, as well as other studies, indicate that the 3,1-TPro mechanism of action is similar to that of other photosensitizers, but may include some minor differences. The conclusion is that 3,1-TPro and other PFP offer a class of effective photosensitizers that may be exploited for their structural versatility, straightforward synthesis leading to a compound of high purity and known structure, and stability (both in terms of shelf-life and in vivo metabolism) as potential candidates for PDT.

Animals↗

PubMed1994

A requirement of hydrophobic and basic amino acid residues for substrate recognition by Ca2+/calmodulin-dependent protein kinase Ia.

The substrate recognition determinants of Ca2+/calmodulin-dependent protein kinase Ia were investigated by using peptide analogues based on the amino acid sequence around Ser-9 of synapsin I. The Km and Vmax for the synthetic peptide Leu-Arg-Arg-Arg-Leu-Ser-Asp-Ala-Asn-Phe are 3.9 microM and 18.5 mumol/(min.mg), respectively. Deletion of Leu at the -5 position lowers the Vmax/Km by 470-fold. The requirement for a hydrophobic residue at -5 was confirmed by the 90- to 2400-fold reduction in Vmax/Km produced by Arg, Ala, or Asp substitutions, but only 2.6-fold decrease after Phe substitution at this position. A hydrophobic residue is similarly required at the +4 position. Deletion of Phe at this position produces a 67-fold reduction, and substitution of Ala for Phe a 43-fold reduction in Vmax/Km. In contrast, substitution with Leu increases Vmax/Km by 1.8-fold. Arg at -3 is also required for recognition as shown by an approximately 240-fold decrease in Vmax/Km after Ala substitution at this position. Positions -2, -4, and +1 appear to play secondary roles in substrate recognition. Arg at -2 and -4 are positive determinants, since Ala substitution at these positions decreases Vmax/Km by 4.7- and 11-fold, respectively. Asp at +1 is a negative influence, since Ala and Leu substitutions at this position increase Vmax/Km by 2.3- and 6.3-fold, respectively. Substitution of Ala for Leu at -1 or Thr for Ser at the 0 position has little effect on phosphorylation kinetics. Thus, Ca2+/calmodulin-dependent protein kinase Ia has the minimal substrate recognition motif of Hyd-Xaa-Arg-Xaa-Xaa-(Ser*/Thr*)-Xaa-Xaa-Xaa-Hyd, where Hyd represents a hydrophobic amino acid residue.

Amino Acid Sequence↗

PubMed1994

Phenol kinase activity of the serine/threonine-specific cAMP-dependent protein kinase: steric and electronic effects.

We have found that the cAMP-dependent protein kinase catalyzes the phosphorylation of a wide variety of peptide-based aromatic alcohols, thereby greatly amplifying the range of compounds recognized as substrates by this enzyme. This newly discovered enzyme-catalyzed reaction is sensitive to both steric and electronic effects. Substituents on the aromatic ring that are positioned para to the hydroxyl moiety lower the observed Km, presumably via a favorable interaction with an adjacent hydrophobic pocket. In contrast, electron-withdrawing substituents have a slight adverse effect on the kinetics of phosphoryl transfer, an observation which is consistent with the notion that the rate of substrate turnover is dependent upon the nucleophilicity of the phosphorylatable hydroxyl moiety. As a corollary, electron-donating groups on the aromatic nucleus promote the rate of phosphoryl transfer to such an extent that the observed Vmax values approach those exhibited by aliphatic alcohols. This suggests that analogously appended electron-donating groups on tyrosine moieties could dramatically improve the modest Vmax values that are typical for tyrosine kinase-catalyzed reactions.

Amino Acid Sequence↗

PubMed1994

The active site substrate specificity of protein kinase C.

The active site substrate specificity of protein kinase C (PKC) has been evaluated. Like the cAMP-dependent protein kinase (PKA), PKC will efficiently phosphorylate achiral residues attached to an active site-directed peptide. In contrast, PKC exhibits behavior that is dramatically different from PKA with respect to the phosphorylation of alpha-substituted alcohols. Although PKA will only phosphorylate residues that contain the same stereochemistry as that found in L-serine, PKC will phosphorylate alpha-configurational isomers that correspond to both the L- and D-stereoisomers. The possible structural basis for the "dual specificity" of PKC is explored. In an analogous vein, although beta-substituted alcohols that serve as PKA substrates must contain the same stereochemistry as that present in L-threonine, PKC will phosphorylate configurational isomers which correspond to both L-threonine and L-allo-threonine. The implications of these observations with respect to protein kinase inhibitor design are discussed.

Amino Acid Sequence↗

PubMed1994

The active site substrate specificity of protein kinase C.

The active site substrate specificity of protein kinase C (PKC) has been evaluated. Like the cAMP-dependent protein kinase (PKA), PKC will efficiently phosphorylate archiral residues attached to an active site-directed peptide. In contrast, PKC exhibits behavior that is dramatically different from PKA with respect to the phosphorylation of alpha-substituted alcohols. Although PKA will only phosphorylate residues that contain the same stereochemistry as that found in L-serine, PKC will phosphorylate alpha-configurational isomers that correspond to both the L- and D-stereoisomers. The possible structural basis for the "dual specificity" of PKC is explored. In an analogous vein, although beta-substituted alcohols that serve as PKA substrates must contain the same stereochemistry as that present in L-threonine, PKC will phosphorylate configurational isomers which correspond to both L-threonine and L-allo-threonine. The implications of these observations with respect to protein kinase inhibitor design are discussed.

Alcohols↗

PubMed1993

Stereochemistry specifies the regiochemistry of phosphorylation in two cAMP-dependent protein kinase substrates.

The substrate specificity of the cAMP-dependent protein kinase has been assessed with peptides bearing threoninol diastereomers. The threoninol residue contains both a primary alcohol and a secondary alcohol, either of which may serve as a site of phosphorylation. The enzyme-catalyzed phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2R,3R)-threoninol furnishes a Km of 498 +/- 39 microM and a Vmax of 7.8 +/- 0.2 mumol/min-mg, whereas the phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2S,3S)-threoninol provides a Km of 16.3 +/- 0.8 microM and a Vmax of 16.0 +/- 0.4 mumol/min-mg. Mass spectral analysis of the phosphopeptide reaction products revealed that each species is phosphorylated only once. 1H-coupled 31P NMR experiments unequivocally demonstrated that the (2R,3R)-isomer is specifically phosphorylated at the secondary alcohol, whereas the (2S,3S)-isomer is exclusively phosphorylated at the primary alcohol. This regiospecificity appears to be a consequence of the stereochemistry at C-2 in the threoninol residues. The structural attributes of the protein kinase that appear to be responsible for the observed differentiation between the C-2 stereoisomers is discussed.

Amino Acid Sequence↗

PubMed1993

Covalent modification with concomitant inactivation of the cAMP-dependent protein kinase by affinity labels containing only L-amino acids.

Affinity labels for proteins that process other proteins (e.g. proteinases and protein kinases) are an amalgam of two components, an active site-directed peptide carrier and a non-peptidic electrophilic appendage. We have synthesized several affinity labels for the cAMP-dependent protein kinase that are composed solely of L-amino acids and therefore contain only functionality present in naturally occurring proteins. We have found that 2 adjacent cysteine residues, when covalently linked via a peptide bond and an intramolecular disulfide loop (abbreviated as Cys<-->Cys), serves as a potent electrophile. The peptides Leu-Arg-Arg-Cys<-->Cys-Leu-Gly (1), Leu-Arg-Arg-Ala-Cys<-->Cys-Gly (2), and Leu-Arg-Arg-Ala-Ala-Cys<-->Cys-Gly (3) inactivate the cAMP-dependent protein kinase in a time-dependent fashion. Since dialysis does not restore activity, but dithiothreitol does, this strongly suggests that covalent modification of the target enzyme has occurred at a cysteine residue. Although there are 2 cysteine moieties contained within the protein kinase, the 14C-acetylated affinity labels modify the enzyme only once. In addition, since ATP blocks inactivation of the protein kinase, this implies that it is the active site cysteine residue (Cys-199) that has undergone covalent modification. Based on the KI(inact) values obtained from inactivation kinetics, we conclude that the optimal site on the affinity label for the electrophilic Cys<-->Cys is 1 amino acid removed from the 2 arginine residues (i.e. 2). In addition, the efficacy of these inhibitors is also dependent upon the size of the disulfide ring. The eight-membered disulfide ring-containing peptides 1-3 are relatively poor affinity labels compared to the 12-membered ring-containing inhibitor, [formula: see text]

Affinity Labels↗

PubMed1993

ATPase-promoting dead end inhibitors of the cAMP-dependent protein kinase.

The cAMP-dependent protein kinase is a bifunctional enzyme, catalyzing the phosphorylation of the serine and threonine residues in peptides and proteins (kinase activity) as well as the phosphorylation of water (ATPase activity). We have found that several peptides, which serve as inhibitors of the kinase reaction, will either maintain or enhance the ATPase reaction catalyzed by the enzyme. Positively charged dipeptides (e.g. Arg-Arg), as well as small guanidino-containing compounds (e.g. guanethidine) block protein kinase activity yet enhance ATPase activity up to 3.5-fold over that exhibited by the enzyme in the absence of these compounds. In contrast, several nonphosphorylatable peptides, whose primary sequences are based on that of a known substrate (i.e. Leu-Arg-Arg-Ala-Ser-Leu-Gly), such as Leu-Arg-Arg-Ala-Ala-Leu-Gly, Leu-Arg-Arg-Ala-Phe-Leu-Gly, and Leu-Arg-Arg-Ala-Tyr-Leu-Gly, have little or no effect on the rate of the kinase-catalyzed hydrolysis of ATP. An exception to the latter observation is Leu-Arg-Arg-Ala-Cys-Leu-Gly, a cysteine-containing peptide that promotes the protein kinase-catalyzed ATPase reaction by 2.2-fold. We have also found that peptides that possess relatively large amino acid side chain moieties immediately following the arginine dyad (i.e. such as Phe, Tyr, Cys, or Asn at Xaa in Leu-Arg-Arg-Xaa-Ala-Leu-Gly) sharply reduce the rate of enzyme-catalyzed ATP hydrolysis. This suggests that in the presence of peptides containing an -Arg-Arg-Ala- sequence, the enzyme-bound gamma-phosphate of ATP is relatively accessible to water. In contrast, when the latter alanine moiety is replaced by a larger residue, access by water to ATP appears to be hindered. These results indicate that certain structural features associated with the substrate or substrate analog have a profound influence on the manner by which these species interact with the protein kinase. Furthermore, the work described herein demonstrates that it is possible to block the physiologically important kinase reaction and simultaneously promote the energetically wasteful ATPase reaction.

Adenosine Triphosphatases↗

PubMed1993

The active site substrate specificity of the cAMP-dependent protein kinase.

cAMP-dependent protein kinase substrates have been synthesized employing an unusually efficient method that allows the alcohol-bearing residue to be incorporated into the peptide after solid phase peptide synthesis. These peptide substrates have been utilized to map the active site substrate specificity of the protein kinase. Only alpha- or beta-substituted alcohol-bearing residues containing the proper absolute configuration are phosphorylated by the enzyme. However, the cAMP-dependent protein kinase will phosphorylate achiral residues. The implications of the observed protein kinase substrate specificity with respect to inhibitor design are discussed.

Alcohols↗

PubMed1992

Physicochemical behavior of cytotoxic ether lipids.

The physicochemical properties of four biologically active alkyllysophosphatidylcholine analogs have been investigated using differential scanning calorimetry (DSC), 31P NMR, and fluorescence spectroscopy. Each of the alkyl ether lipids, suspended in a buffer/ethylene glycol matrix, undergo a thermotropic structural rearrangement at temperatures near 0 degree C. The axially asymmetric 31P NMR spectra obtained for all four lipids, after prolonged incubation at -20 degrees C, are consistent with the notion that these amphiphiles are able to form extended lamellar structures at temperatures below the main phase transition temperature (Tm). We have attributed the sharp, isotropic 31P resonance, observed for each derivative at temperatures above the Tm, to the presence of a micellar state. This has been confirmed via 31P NMR experiments employing the lanthanide shift reagent PrCl3. The critical micelle concentrations (cmc) of these lysolipids have been determined via the fluorescence titration of [6-palmitoyl-2-[[(2-trimethylammonium)ethyl]methyl]amino] naphthalene (PATMAN). The low values obtained for both the lamellar to micellar phase transition temperature (-7.88 +/- 0.19 degree C) and the cmc (0.15 +/- 0.02 microM) for the 2-benzyloxylysolipid (1) indicate that this analog exhibits a marked preference for the micellar state relative to the 2-methoxy (2), 2-deoxy (3), and 2-hydroxy (4) substituted compounds.

Calorimetry, Differential Scanning↗

PubMed1992

NMR spectral analysis of cytotoxic ether lipids.

Several cytotoxic ether lipid analogs of platelet activating factor exhibit a wide range of interesting pharmacological properties. Furthermore, at least two members of this family of lipids have progressed to phase I clinical trials as potential cancer chemotherapeutic agents. In spite of the promise that these compounds hold as anticancer drugs, they remain poorly characterized. We report herein the first complete 1H NMR analysis of several palmityl-based ether lipids. In addition, we report the 13C NMR spectral assignments for these lipids, which are based, in part, on both the presence and magnitude of 31P-13C and 14N-13C coupling constants.

Antineoplastic Agents↗

PubMed1990

Noncovalent active site interactions enhance the affinity and control the binding order of reversible inhibitors of the cAMP-dependent protein kinase.

The peptides, Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH2, Leu-Arg-Arg-Gln-Ala-Leu-Gly-NH2, and Leu-Arg-Arg-Asn-Ala-Leu-Gly-NH2, serve as active site-directed inhibitors of the cAMP-dependent protein kinase from bovine cardiac muscle. The Asn-containing peptide is a 10-fold more potent inhibitor than its Ala- and Gln-containing counterparts. All three peptides are linear competitive inhibitors versus a peptide-based substrate and uncompetitive inhibitors versus MgATP. The enhanced inhibitory potency of the Asn-peptide, in conjunction with the observed loss of ATP-ase activity of the enzyme in the presence of the inhibitor, suggests that asparagine may serve as a through-space isostere of serine. The uncompetitive inhibition pattern displayed by amide-capped peptides versus MgATP indicates that these species bind in an ordered fashion to the cAMP-dependent protein kinase, with MgATP binding first.

Adenosine Triphosphatases↗

PubMed1989

Multiple arginine residues contribute to the increased efficacy of peptide substrates for the cAMP-dependent protein kinase.

Efficient cAMP-dependent protein kinase substrates typically contain an arginine dyad one amino acid removed from the residue which undergoes phosphorylation (ie. Arg-Arg-X-Ser). However, several naturally occurring protein kinase inhibitors and substrates possess additional basic residues that are proximal to the arginine dyad, implying the presence of either an extended or an additional acidic subsite on the enzyme. In this study, we investigated the substrate efficacy of several multiple arginine-bearing peptides. The most efficient substrate studied, Arg-Arg-Leu-Arg-Arg-Ala-Ser-Leu-Gly, exhibits a nearly eleven-fold increase in kcat/Km relative to Leu-Arg-Arg-Ala-Ser-Leu-Gly. The enhanced kcat/Km is primarily a consequence of a reduced Km. These results suggest that a double arginine dyad, separated by a single amino acid, represents the optimal sequence for basic residues on cAMP-dependent protein kinase substrates.

Amino Acid Sequence↗

PubMed1989

The cyclic AMP-dependent protein kinase from bovine cardiac muscle is a homoserine kinase.

The catalytic subunit of the cAMP-dependent protein kinase from bovine cardiac muscle phosphorylates homoserine in the synthetic peptide Leu-Arg-Arg-Ala-Hse-Leu-Gly. Phosphorylation of the primary alcohol of the homoserine residue was established via NMR spectroscopy. Two-dimensional correlated and nuclear Overhauser effect spectroscopies provided the sequence-specific chemical shift assignments of the substrate peptide and its phosphorylated counterpart. Coupled and decoupled 31P NMR experiments established the presence of phosphate on the homoserine residue. The maximal velocity (6.4 mumol/min.mg) obtained for homoserine-peptide phosphorylation at 12.5 mM Mg2+ compares favorably to the velocities observed for the corresponding serine- (21 mumol/min.mg), threonine- (3.2 mumol/min.mg), and hydroxyproline-peptides (1 mumol/min.mg). However, the Km for homoserine kinase activity is modest (1.3 mM) relative to the Km associated with the phosphorylation of the serine-containing substrate (22 microM). The effect of Mg2+ concentration on the kinetic parameters kcat, Km, and kcat/Km was investigated for both serine- and homoserine-peptides. Both substrates display similar kcat/Km versus [Mg2+] profiles, with the most notable difference that the optimal Mg2+ concentration is higher for the homoserine-containing peptide. In addition, the Km for the serine-peptide was found to be independent of [Mg2+], whereas the Km for the homoserine-peptide was observed to be dependent upon [Mg2+]. These results suggest that the long homoserine side chain may induce an unusually large off rate for the peptide and/or may misalign the hydroxyl moiety in the active site.

Amino Acid Sequence↗

PubMed1989

Intrasubstrate steric interactions in the active site control the specificity of the cAMP-dependent protein kinase.

The cAMP-dependent protein kinase catalytic subunit phosphorylates serine residues more efficiently than threonine residues in synthetic peptides. In marked contrast, both amino acids are phosphorylated at similar rates when contained within the appropriate intact protein substrate. The structural basis for the discriminatory behavior observed in small peptides has been investigated and found to be a result of intrapeptide steric interactions in the vicinity of the threonine alcohol moiety. Leu-Arg-Arg-Gly-Thr-Leu-Gly, which is nearly free of these interactions, is phosphorylated at a rate that is almost comparable to its serine-containing counterpart.

Amino Acid Sequence↗

PubMed1989

Cryopreservation of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase from bovine cardiac muscle.

A method for the cryogenic storage of the cAMP-dependent protein kinase from bovine cardiac muscle is described. The catalytic parameters, kcat, KM, and kcat/KM are used to assess the activity of the enzyme both prior and subsequent to the freeze-thaw cycle. The enzyme is stored in cryogenic vials at -196 degrees C in liquid nitrogen. Complete retention of catalytic activity is dependent upon a rapid and efficient freeze-thaw cycle and the use of morpholinepropanesulfonic acid as the buffer. In addition, this buffer appears to eliminate the KCl- or NaCl-induced damage typically observed for enzymes stored at low temperature in phosphate buffer. As a result, morpholinepropanesulfonic acid may prove to be a more appropriate cryopreservation buffer than phosphate when the presence of salt is required for enzyme solubility or stability.

Animals↗

PubMed1985

The chemical modification of enzymatic specificity.

The chemical modification of enzymes has played and will continue to play an important role in probing the mechanism of enzyme activity. This technique can be utilized for identification of those individual amino acid residues responsible for the catalytic properties of the entire protein. In chemical modification experiments, changes in enzymatic specificity have been noted, but often not predicted. However, in recent years, rational approaches for the alteration of enzymatic properties have become feasible by means of site-specific mutagenesis and chemical methodology. In the first method, one amino acid can be replaced by a new one; in the second method, not only can new amino acid residues be introduced but also new catalytic entities (such as flavins) can be affixed to the protein molecule. Both methodologies are in their infancy, yet they represent a potentially powerful approach toward the design and synthesis of enzymes possessing new specificities.

Allosteric Regulation↗