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E T Kaiser

Publications and source records attributed to E T Kaiser.

At least 37 records · Page 2Linked to original sources
PubMed1988

Stereochemistry of phospho group transfer catalyzed by a mutant alkaline phosphatase.

The stereochemical course of the phospho group transfer catalyzed by mutant (S102C) alkaline phosphatase from Escherichia coli was investigated by using 31P nuclear magnetic resonance spectroscopy. Transphosphorylation from 4-nitrophenyl (Rp)-[16O, 17O, 18O]phosphate to (S)-propane-1,2-diol occurs with overall retention of configuration at phosphorus. This result is consistent with the view that the hydrolysis of substrates by this mutant enzyme proceeds by way of a covalent phosphoenzyme intermediate in the same manner as the wild-type alkaline phosphatase.

Alkaline Phosphatase↗
PubMed1988

A functional decaisoleucine-containing signal sequence. Construction by cassette mutagenesis.

An alkaline phosphatase signal sequence optimized for formation of a hydrophobic alpha-helix functions very efficiently in the transport process. This mutant contained a core region comprised of 9 consecutive leucine residues (Kendall, D. A., Bock, S. C., and Kaiser, E. T. (1986) Nature 321, 706-708). We have now constructed a second mutant containing a decaisoleucine core region. Isoleucine was chosen because it is an isomer of leucine with comparable hydrophobicity but in synthetic peptides isoleucine favors beta-sheet formation. Surprisingly, this mutant precursor was also processed efficiently, and mature alkaline phosphatase was correctly targeted to the Escherichia coli periplasm. Since the effective length of a beta-strand is extended relative to an alpha-helix, conformational differences should be mirrored by the relative effectiveness of shortened polyisoleucine and polyleucine core regions. However, analysis of two additional mutants containing truncated segments of either polyleucine or polyisoleucine did not reveal any differences and both accumulate as precursors. We conclude that these mutants do not adopt critically different structures. This comparative analysis was facilitated by construction of a new plasmid, CASS3. This plasmid contains unique restriction sites flanking the DNA region coding for the signal sequence hydrophobic core segment. Consequently, the wild type core-encoding region can be readily replaced with synthetic oligonucleotides coding for new structural units and multiple amino acid substitutions can be made without the need for step-wise mutagenesis.

Alkaline Phosphatase↗
PubMed1988

Identification of amino acid residues involved in substrate recognition by the catalytic subunit of bovine cyclic AMP dependent protein kinase: peptide-based affinity labels.

Two peptide-based affinity inactivators Ac-Leu-(BrAc)Orn-Arg-Ala-Ser-Leu-Gly (4) and Ac-Leu-Arg-(BrAc)Orn-Ala-Ser-Leu-Gly (5) were prepared as probes for the study of the nature of the active-site residues in the catalytic subunit of cyclic AMP dependent protein kinase. Under conditions of inhibitor in excess, both peptides inactivated the catalytic subunit by an apparent biphasic process. A fast phase, which inactivated the protein by approximately 40%, was followed by a slow phase that accounted for the loss of the remaining enzyme activity. Protection experiments with the kinase substrates showed that the slow phase of inactivation was active site directed, while the fast phase was not. Studies with radioactively labeled peptides 4 and 5 indicated incorporation of two peptide residues per molecule of the catalytic subunit upon complete inactivation. This observation is consistent with the occurrence of one alkylation event in each phase of the inactivation. The protein was proteolyzed subsequent to its modification with radioactive peptides. High-performance liquid chromatography afforded two radioactive peptide fragments in each case, which were sequenced by Edman degradation. Peptide 4 alkylated Thr-197 and Glu-346, while peptide 5 modified Cys-199 and also Glu-346. Data are presented to support the conclusion that Thr-197 and Cys-199 are located at or near the active site.

Affinity Labels↗
PubMed1988

Use of site-directed mutagenesis to elucidate the role of arginine-166 in the catalytic mechanism of alkaline phosphatase.

The guanidinium group of arginine-166 has been postulated to act as an electrophilic species during phosphorylation of alkaline phosphatase. Its role could be either to stabilize the developing negative charge on the oxygen of the leaving group or the pentacoordinate transition state or to help bind the -PO2-3 group. We have produced via site-directed mutagenesis two Escherichia coli alkaline phosphatase mutants (lysine-166 and glutamine-166) to test whether the guanidinium group plays a critical role in catalysis. Comparative kinetic characterization of the lysine-166 and glutamine-166 mutants indicates that the charge at residue 166 is not required for the hydrolysis of phosphate monoesters. Small decreases in kcat are observed for both the lysine and glutamine mutants, relative to the wild-type enzyme, but the value for the uncharged glutamine mutant is only one-third that of lysine. Thus, the stabilizing effect of the positively charged guanidinium group does not appear to play a major role in the rate-limiting step for substrate hydrolysis. A significant effect on the Km value is seen only for the glutamine mutant.

Alkaline Phosphatase↗
PubMed1988

Probing the peptide binding site of the cAMP-dependent protein kinase by using a peptide-based photoaffinity label.

A peptide-based photoaffinity label for the catalytic subunit of the cAMP-dependent protein kinase was prepared from the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)]. By using solid-phase peptide synthesis methodology, DL-Phe(pBz) was incorporated into the cAMP-dependent protein kinase substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly in place of the phosphorylatable serine. The diastereomeric peptides were separated by reverse-phase HPLC. The peptide substrate analog containing L-Phe(pBz) had a Ki of approximately 110 microM at pH 7.5. When photolyzed at 350 nm in the presence of the enzyme, this peptide caused time- and concentration-dependent inactivation. Radioactive acetylated L-Phe(pBz) peptide was used to establish the binding stoichiometry of peptide to enzyme; these results, together with protection experiments, showed the photoaffinity labeling to be specific (approximately 1:1). To identify the residues that were modified on the catalytic subunit, the photoinactivated enzyme was cleaved with CNBr and V8 protease (Staphylococcus aureus). The resulting peptide fragments were purified by HPLC and were sequenced; these experiments identified the modified residues as Gly-125 and Met-127. This region of the cAMP-dependent protein kinase catalytic subunit contains many residues that are conserved in serine- and tyrosine-protein kinases.

Affinity Labels↗
PubMed1988

Synthesis and biological evaluation of pNPY fragments.

Peptide fragments of pNPY corresponding to the C-terminal segments (13-36) and (25-36), the N-terminal segments (1-12) and (1-24), the segments (6-14) and (7-20), which contain a putative beta-turn, and the internal segments (13-24) and (20-30) were synthesized using solid phase methodology. These fragments were assayed for NPY receptor binding activity in the rat hypothalamus membrane preparation, enhancement of food intake in the rat following ivt administration and inhibition of electrically stimulated muscle contraction in the rat vas deferens. Only the C-terminal fragment (13-36) retained some of the activities of pNPY, appearing to act as a weak agonist, having an additive effect with pNPY on the inhibition of muscle contraction and prolonging the duration of action of pNPY in the feeding assay. It also had considerable alpha-helical character, as did pNPY. None of the other peptide fragments had any agonist or antagonist activity. These results suggest that the expression of full biological NPY activity requires both the C- and the N-terminal segments as well as a putative amphiphilic alpha-helical segment (14-31).

Amino Acid Sequence↗
PubMed1987

Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities.

As part of a search for peptides that have specificity for selected protein kinases, the possibility that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) recognizes the hydrogen-bonding potential of its peptide substrates was investigated. A-Kinase catalyzes the phosphorylation of five N alpha-methylated and four depsipeptide derivatives of Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) at rates that differ by at least 7 orders of magnitude. These peptide 1 analogues each lack the ability to donate a hydrogen bond at selected positions in the peptide chain. If a particular amide hydrogen of a peptide amide is involved in hydrogen bonding, which is important for enzyme recognition, the prediction is that peptides which contain an ester or a N-methylated bond at that position in peptide 1 will be comparatively poor substrates. In contrast, if a depsipeptide has a reactivity comparable to that of peptide 1 but the analogous N-methylated peptide has a poor reactivity with A-kinase, the result might indicate that the N-methyl group causes unfavorable steric effects. The depsipeptide that lacks a Leu6 amide proton is a good substrate for A-kinase, but the corresponding N-methylated peptide is phosphorylated far less efficiently. This result and others presented in this paper suggest that although enzyme-substrate hydrogen bonding may play some role in A-kinase catalysis of phosphoryl group transfer, other explanations are necessary to account for the relative reactivities of N alpha-methylated and depsi-containing peptide 1 analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Conformation of Leu-Arg-Arg-Ala-Ser-Leu-Gly bound in the active site of adenosine cyclic 3',5'-phosphate dependent protein kinase.

Studies utilizing NMR spectroscopy have shown that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) probably binds Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) in one of two extended coil conformations (A or B). The relative reactivities of a series of N-methylated peptides based on the structure of peptide 1 might, therefore, be related to how well each can assume the A or B conformation. From estimates of the magnitude of steric interactions that would be induced by N-methylation of an amide in peptide 1 that is locked in either conformation, the ability of each peptide to form that conformation was predicted. The ability of A-kinase to catalyze phosphorylation of the N-methylated peptides correlated well with the ability of each peptide to form conformation A, but not conformation B. In accord with these findings, the reactivity of an unreactive N-methylated peptide was partially restored by a second change, which allowed the peptide to assume conformation A. These results suggest that, when bound in the enzymatic active site, peptide 1 has a conformation that resembles structure A much more closely than structure B.

Amino Acid Sequence↗
PubMed1987

Distinguishing among protein kinases by substrate specificities.

In the previous paper, N-methylated peptides were shown to be sensitive probes of substrate conformation within the adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) active site. While it has been shown that other protein kinases will catalyze the phosphorylation of the same peptide sequences as A-kinase, there is as yet little information as to whether the protein kinases differentiate between substrates on the basis of conformation. For this reason, the conformationally restricted N-methylated peptides were used to probe the active site of guanosine cyclic 3',5'-phosphate dependent protein kinase (G-kinase), which is homologous in sequence to [Takio, K., Wade, R. D., Smith, S. B., Krebs, E. G., Walsh, K. A., & Titani, K. (1984) Biochemistry 23, 4207-4218] and which has substrate specificities similar to [Lincoln, T. M., & Corbin, J. D. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3239-3243] those of A-kinase. Although this enzyme appears to bind the peptides in a conformation resembling that of conformation A, it is more able to accommodate backbone methylation than is A-kinase. A peptide substrate at least 700-fold selective for G-kinase over A-kinase was found. Backbone methylation may, therefore, represent a way of making peptide substrates and inhibitors selective for a particular kinase.

Animals↗
PubMed1987

Use of directed mutagenesis to probe the role of tyrosine 198 in the catalytic mechanism of carboxypeptidase A.

Derivatization of Tyr198 in carboxypeptidase A (CPA) results in lowered catalytic activity toward peptide substrates (Cueni, L., and Riordan, J.F. (1978) Biochemistry 17, 1834-1842). We have synthesized via directed mutagenesis a rat CPA variant [Phe198] CPA containing a Tyr198-to-Phe substitution in order to test whether the phenolic hydroxyl plays a critical role in catalysis. A double mutant [Phe193, Phe248]CPA in which both Tyr198 and Tyr248 have been replaced by phenylalanine has also been engineered. Enzymatic characterization of [Phe198]CPA indicates that the Tyr198 hydroxyl is not obligatory for the hydrolysis of peptide and ester substrates. Furthermore, parallel studies with [Phe198, Phe248]CPA show that simultaneous removal of both the Tyr198 and Tyr248 hydroxyls does not abolish catalytic activity. Analysis of the acetylated derivatives of [Phe198]CPA, [Phe248]CPA, and [Phe198, Phe248]CPA establishes that Tyr198 and Tyr248 are the active site tyrosines which are modified by N-acetylimidazole. In addition, the perturbations of enzymatic activity which accompany acetylation of native CPA can be largely assigned to derivatization of Tyr248. The changes in the kinetic constants of substrate hydrolysis due to the Tyr198-to-Phe substitution are manifested as small decreases in the kcat values, but the Km values are essentially unaffected. This exclusive effect on the kcat values suggests that the Tyr198 hydroxyl participates in catalysis by stabilizing the rate-determining transition-state complex.

Amino Acid Sequence↗
PubMed1987

Biological and physical properties of a model calcitonin containing a glutamate residue interrupting the hydrophobic face of the idealized amphiphilic alpha-helical region.

2A new calcitonin analogue, model calcitonin III (MCt-III), has been synthesized, and its biological and physical characteristics have been studied. This analogue has an idealized alpha-helix from residue 8-22 with glutamate at position 15 interrupting an otherwise continuous surface of aliphatic side chains (those of leucine residues) on the hydrophobic face of the helix. MCt-III differs from a previous model, MCt-II, only by the substitution Leu15----Glu and is here compared with salmon calcitonin I (sCt-I) and MCt-II to elucidate further the role of the putative amphiphilic alpha-helix in determining biological and physical properties of the hormone. MCt-III shows physical properties intermediate between those of sCt-I and MCt-II, demonstrating the influence of appropriately positioned single residues on properties of amphiphilic structures. In our two biological assays, a brain-binding assay and an in vivo hypocalcemic assay, MCt-III reproduces the sigmoidal dose-response curves of sCt-I; this contrasts with the behavior of MCt-II, which demonstrated unusual dose-response curves in these two assays. MCt-III is almost three times more potent than sCt-I in our hypocalcemic assay; this activity groups MCt-III among the most potent known analogues of sCt-I.

Animals↗
PubMed1986

Design of biologically active peptides with non-peptidic structural elements. Biological and physical properties of a synthetic analogue of beta-endorphin with unnatural amino acids in the region 6-12.

Modelling studies with beta-endorphin have clearly demonstrated that an amphiphilic secondary structural segment is a salient feature of the biologically active conformation of this 31-residue opioid peptide hormone. Here, we have initiated the synthesis of peptide models using unnatural building blocks by designing a beta-endorphin analogue (peptide 6) in which the hydrophilic linker region between the NH2-terminal enkephalin (residues 1-5) and the COOH-terminal helix (residues 10-28, sequence identical to that of peptide 3 in region 13-31, Fig. 1) consists of four units of gamma-amino-gamma-hydroxymethylbutyric acid connected by isopeptidic linkages. Peptide 6 has physical properties similar to that of peptide 3, as shown by surface monolayer and circular dichroism studies. The binding affinities of the two peptides to delta- and mu-receptors are also similar. In rat vas deferens assays, the present model is equipotent to peptide 3. The most striking result of all is the potent analgesic activity displayed by peptide 6 when injected intracerebroventricularly into mice. The potencies of peptides 6 and 3 are comparable in these assays. These studies clearly illustrate that one can use unusual building blocks to construct structural regions of synthetic analogues and still preserve the biological activity of peptide hormones.

Analgesics↗