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PubMed1997

Point mutations at the purine nucleoside phosphorylase locus impair thymocyte differentiation in the mouse.

Three point mutations on the Np(b) allele of the purine nucleoside phosphorylase locus in the mouse have been recovered by male germ cell mutagenesis. The mutants were backcrossed, 12-14 generations, and are designated in increasing order of severity of enzyme deficiency and phenotype: B6-NPE, Met-87 --> Lys; B6-NPF, Ala-228 --> Thr; and B6-NPG, Trp-16 --> Arg. A marked decline in total cell numbers per thymus occurs between 2 and 3 months for the more severe B6-NPF and B6-NPG mutants (35% and 52%, respectively) and by 8 months for the less severe B6-NPE mutation. The thymocyte population is thereafter characterized by a 3- or 8-fold expanded precursor, CD4-CD8- double-negative population and 15% or 55% reduced CD4+CD8+ double-positive cells for the B6-NPF and B6-NPG strains, respectively. Spleen lymphocyte Thy-1+ cells are reduced by 50% and spleen lymphocyte response to T cell mitogen and interleukin 2 is reduced by 80%. Increases of thymocyte dGTP pools of 5- and 2.5-fold for B6-NPF and B6-NPG mutants, respectively, are observed. The purine nucleoside phosphorylase-deficient mouse exhibits age-dependent progressive perturbations in thymocyte differentiation, reduced numbers of thymocytes, and reduced splenic T cell numbers and response. The progressive T cell deficit is similar to the human disorder.

Alleles↗
PubMed1998

Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo.

An inhibitor of human liver glycogen phosphorylase a (HLGPa) has been identified and characterized in vitro and in vivo. This substance, [R-(R*, S*)]-5-chloro-N-[3-(dimethylamino)-2-hydroxy-3-oxo-1-(phenylmethyl)pr opyl]-1H-indole-2-carboxamide (CP-91149), inhibited HLGPa with an IC50 of 0.13 microM in the presence of 7.5 mM glucose. CP-91149 resembles caffeine, a known allosteric phosphorylase inhibitor, in that it is 5- to 10-fold less potent in the absence of glucose. Further analysis, however, suggests that CP-91149 and caffeine are kinetically distinct. Functionally, CP-91149 inhibited glucagon-stimulated glycogenolysis in isolated rat hepatocytes (P < 0.05 at 10-100 microM) and in primary human hepatocytes (2.1 microM IC50). In vivo, oral administration of CP-91149 to diabetic ob/ob mice at 25-50 mg/kg resulted in rapid (3 h) glucose lowering by 100-120 mg/dl (P < 0.001) without producing hypoglycemia. Further, CP-91149 treatment did not lower glucose levels in normoglycemic, nondiabetic mice. In ob/ob mice pretreated with 14C-glucose to label liver glycogen, CP-91149 administration reduced 14C-glycogen breakdown, confirming that glucose lowering resulted from inhibition of glycogenolysis in vivo. These findings support the use of CP-91149 in investigating glycogenolytic versus gluconeogenic flux in hepatic glucose production, and they demonstrate that glycogenolysis inhibitors may be useful in the treatment of type 2 diabetes.

Amides↗
PubMed1995

Chimeric muscle and brain glycogen phosphorylases define protein domains governing isozyme-specific responses to allosteric activation.

Muscle and brain glycogen phosphorylases differ in their responses to activation by phosphorylation and AMP. The muscle isozyme is potently activated by either phosphorylation or AMP. In contrast, the brain isozyme is poorly activated by phosphorylation and its phosphorylated a form is more sensitive to AMP activation when enzyme activity is measured in substrate concentrations and temperatures encountered in the brain. The nonphosphorylated b form of the brain isozyme also differs from the muscle isozyme b form in its stronger affinity and lack of cooperativity for AMP. To identify the structural determinants involved, six enzyme forms, including four chimeric enzymes containing exchanges in amino acid residues 1-88, 89-499, and 500-842 (C terminus), were constructed from rabbit muscle and human brain phosphorylase cDNAs, expressed in Escherichia coli, and purified. Kinetic analysis of the b forms indicated that the brain isozyme amino acid 1-88 and 89-499 regions each contribute in an additive fashion to the formation of an AMP site with higher intrinsic affinity but weakened cooperativity, while the same regions of the muscle isozyme each contribute to greater allosteric coupling but weaker AMP affinity. Kinetic analysis of the a forms indicated that the amino acid 89-499 region correlated with the reduced response of the brain isozyme to activation by phosphorylation and the resultant increased sensitivity of the a form to activation by saturating levels of AMP. This isozyme-specific response also correlated with the glycogen affinity of the a forms. Enzymes containing the brain isozyme amino acid 89-499 region exhibited markedly reduced glycogen affinities in the absence of AMP compared to enzymes containing the corresponding muscle isozyme region. Additionally, AMP led to greater increases in glycogen affinity of the former set of enzymes. In contrast, phosphate affinities of all a forms were similar in the absence of AMP and increased approximately the same extent in AMP. The potential importance of a number of isozyme-specific substitutions in these sequence regions is discussed.

Adenosine Monophosphate↗
PubMed1995

Characterization of the regulatory domain of the gamma-subunit of phosphorylase kinase. The two noncontiguous calmodulin-binding subdomains are also autoinhibitory.

Phosphorylase kinase is a multimeric protein kinase (alpha 4 beta 4 gamma 4 delta 4) whose enzymatic activity is conferred by its gamma-subunit. A library of 18 overlapping synthetic peptides spanning residues 277-386 of the gamma-subunit has been prepared to use in identifying important regulatory structures in the protein. In the present study, the library was screened to identify regions that might function as autoinhibitory domains. Peptides from two distinct regions were found to inhibit the Ca2(+)-activated holoenzyme. The same regions were previously found to bind calmodulin (i.e. the delta-subunit; Dasgupta, M. Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163). The most potent substrate antagonist peptides were PhK13 (residues 302-326; Ki = 300 nM) and PhK5 (residues 342-366; Ki = 20 microM). Both peptides inhibited the holoenzyme competitively with respect to phosphorylase b and noncompetitively with respect to Mg.ATP. When the pattern of inhibition with both peptides present was analyzed, inhibition was observed to be synergistic and modestly cooperative indicating that the two peptides can simultaneously occupy the protein substrate-binding site(s). These data are consistent with a model in which the regions of the gamma-subunit represented by PhK5 and PhK13 work in concert as regulatory subdomains that transduce Ca2(+)-induced conformational changes in the delta-subunit to the catalytic gamma-subunit through a pseudosubstrate autoinhibitory mechanism.

Amino Acid Sequence↗
PubMed1996

The testis isoform of the phosphorylase kinase catalytic subunit (PhK-gammaT) plays a critical role in regulation of glycogen mobilization in developing lung.

In order to identify the form of phosphorylase kinase catalytic subunit expressed in developing lung, degenerate polymerase chain reaction primers were designed based on conserved domains of the two known catalytic subunits, expressed primarily in muscle and testis. Amplification of cDNA from day 19 fetal rat lung followed by cloning and sequence analyses indicated that only the testis isoform of phosphorylase kinase (PhK-gammaT) was detectable in fetal lung. In situ hybridization analyses indicated that expression of PhK-gammaT RNA in developing lung tissue was widespread and not restricted to Type II epithelial cells; PhK-gammaT protein expression was temporally and spatially correlated with expression of PhK-gammaT RNA. PhK-gammaT RNA and protein expression was also characterized in the PhK-deficient glycogen storage disease (gsd) rat. PhK-gammaT RNA levels were similar in Type II cells isolated from wild type and gsd/gsd fetuses; in contrast, PhK-gammaT protein was virtually undetectable in gsd/ gsd Type II cells and enzyme activity was very low. These results suggest that PhK-gammaT plays a critical role in mobilization of glycogen during fetal lung development and that failure to catabolize glycogen in the gsd/gsd rat is related to an untranslatable PhK-gammaT RNA or unstable protein.

Amino Acid Sequence↗
PubMed1997

High affinity binding and allosteric regulation of Escherichia coli glycogen phosphorylase by the histidine phosphocarrier protein, HPr.

The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts of Escherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP). The high affinity (association constant approximately 10(8) M-1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis. Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase. HPr binding increases GP activity to 250% of the level in control assays. Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the Km for glycogen and an increase in the Vmax for phosphate, indicating a mixed type activation. The stimulatory effect of E. coli HPr on E. coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form. Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important. This novel mechanism for the regulation of GP provides the first evidence directly linking E. coli HPr to the regulation of carbohydrate metabolism.

Allosteric Regulation↗
PubMed2001

Uridine phosphorylase association with vimentin. Intracellular distribution and localization.

Uridine phosphorylase (UPase), a key enzyme in the pyrimidine salvage pathway, is associated with the intermediate filament protein vimentin, in NIH 3T3 fibroblasts and colon 26 cells. Affinity chromatography was utilized to purify UPase from colon 26 and NIH 3T3 cells using the uridine phosphorylase inhibitor 5'-amino benzylacyclouridine linked to an agarose matrix. Vimentin copurification with UPase was confirmed using both Western blot analysis and MALDI-MS methods. Separation of cytosolic proteins using gel filtration chromatography yields a high molecular weight complex containing UPase and vimentin. Purified recombinant UPase and recombinant vimentin were shown to bind in vitro with an affinity of 120 pm and a stoichiometry of 1:2. Immunofluorescence techniques confirm that UPase is associated with vimentin in both NIH 3T3 and colon 26 cells and that depolymerization of the microtubule system using nocodazole results in UPase remaining associated with the collapsed intermediate filament, vimentin. Our data demonstrate that UPase is associated with both the soluble and insoluble pools of vimentin. Approximately 60-70% of the total UPase exists in the cytosol as a soluble protein. Sequential extraction of NIH 3T3 or colon 26 cells liberates an additional 30-40% UPase activity associated with a detergent extractable fraction. All pools of UPase have been shown to possess enzymatic activity. We demonstrate for the first time that UPase is associated with vimentin and the existence of an enzymatically active cytoskeleton-associated UPase.

3T3 Cells↗
PubMed2001

Three-dimensional structure of a hyperthermophilic 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus.

The structure of 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) has been determined alone, as ternary complexes with sulfate plus substrates 5'-deoxy-5'-methylthioadenosine, adenosine, or guanosine, or with the noncleavable substrate analog Formycin B and as binary complexes with phosphate or sulfate alone. The structure of unliganded SsMTAP was refined at 2.5-A resolution and the structures of the complexes were refined at resolutions ranging from 1.6 to 2.0 A. SsMTAP is unusual both for its broad substrate specificity and for its extreme thermal stability. The hexameric structure of SsMTAP is similar to that of purine-nucleoside phosphorylase (PNP) from Escherichia coli, however, only SsMTAP accepts 5'-deoxy-5'-methylthioadenosine as a substrate. The active site of SsMTAP is similar to that of E. coli PNP with 13 of 18 nearest residues being identical. The main differences are at Thr(89), which corresponds to serine in E. coli PNP, and Glu(163), which corresponds to proline in E. coli PNP. In addition, a water molecule is found near the purine N-7 position in the guanosine complex of SsMTAP. Thr(89) is near the 5'-position of the nucleoside and may account for the ability of SsMTAP to accept either hydrophobic or hydrophilic substituents in that position. Unlike E. coli PNP, the structures of SsMTAP reveal a substrate-induced conformational change involving Glu(163). This residue is located at the interface between subunits and swings in toward the active site upon nucleoside binding. The high-resolution structures of SsMTAP suggest that the transition state is stabilized in different ways for 6-amino versus 6-oxo substrates. SsMTAP has optimal activity at 120 degrees C and retains full activity after 2 h at 100 degrees C. Examination of the three-dimensional structure of SsMTAP suggests that unlike most thermophilic enzymes, disulfide linkages play a key in role in its thermal stability.

Binding Sites↗
PubMed2001

Transition state analogue inhibitors of purine nucleoside phosphorylase from Plasmodium falciparum.

Immucillins are logically designed transition-state analogue inhibitors of mammalian purine nucleoside phosphorylase (PNP) that induce purine-less death of Plasmodium falciparum in cultured erythrocytes (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226-3231). PNP is present at high levels in human erythrocytes and in P. falciparum, but the Plasmodium enzyme has not been characterized. A search of the P. falciparum genome data base yielded an open reading frame similar to the PNP from Escherichia coli. PNP from P. falciparum (P. falciparum PNP) was cloned, overexpressed in E. coli, purified, and characterized. The primary amino acid sequence has 26% identity with E. coli PNP, has 20% identity with human PNP, and is phylogenetically unique among known PNPs with equal genetic distance between PNPs and uridine phosphorylases. Recombinant P. falciparum PNP is catalytically active for inosine and guanosine but is less active for uridine. The immucillins are powerful inhibitors of P. falciparum PNP. Immucillin-H is a slow onset tight binding inhibitor with a K(i)* value of 0.6 nm. Eight related immucillins are also powerful inhibitors with dissociation constants from 0.9 to 20 nm. The K(m)/K(i)* value for immucillin-H is 9000, making this inhibitor the most powerful yet reported for P. falciparum PNP. The PNP from P. falciparum differs from the human enzyme by a lower K(m) for inosine, decreased preference for deoxyguanosine, and reduced affinity for the immucillins, with the exception of 5'-deoxy-immucillin-H. These properties of P. falciparum PNP are consistent with a metabolic role in purine salvage and provide an explanation for the antibiotic effect of the immucillins on P. falciparum cultured in human erythrocytes.

Amino Acid Sequence↗
PubMed2006

Thymidine phosphorylase from Escherichia coli: tight-binding inhibitors as enzyme active-site titrants.

Thymidine phosphorylase (EC 2.4.2.4) catalyses the reversible phosphorolysis of pyrimidine 2'-deoxynucleosides, forming 2-deoxyribose-1-phosphate and pyrimidine. 5-Chloro-6-(2-imino-pyrrolidin-1-yl)methyl-uracil hydrochloride (TPI, 1) and its 5-bromo analogue (2), 6-(2-amino-imidazol-1-yl)methyl-5-bromo-uracil (3) and its 5-chloro analogue (4) act as tight-binding stoichiometric inhibitors of recombinant E. coli thymidine phosphorylase, and thus can be used as the first active-site titrants for it using either thymidine or 5-nitro-2'-deoxyuridine as substrate.

Binding Sites↗
PubMed2006

A kinetic, modeling and mechanistic re-analysis of thymidine phosphorylase and some related enzymes.

Thymidine phosphorylase (TP) is an important target enzyme for cancer chemotherapy but currently available inhibitors lack in vivo potency. Related enzymes also are therapeutic targets. A greater understanding of enzyme structure and mechanism may help in the design of improved drugs and this work assists in that regard. Also important is the correct identification of the ionization states and tautomeric forms of substrates and products when bound to the enzyme and during the course of the reaction. Approximate methods for estimating some deltapK(a)s between aqueous and protein-bound substrates are exemplified for nucleobases and nucleosides. The estimates demonstrate that carbonyl-protonated thymidine and hydroxy tautomers of thymine are not involved in TP's actions. Other estimates indicate that purine nucleoside phosphorylase binds inosine and guanosine as zwitterionic tautomers and that phosphorolysis proceeds through these forms. Extensive molecular modeling based on an X-ray structure of human TP indicates that TP is likely to be mechanistically similar to all other natural members of the class in proceeding through a alpha-oxacarbenium-like transition state or states.

Animals↗
PubMed1997

Behavior of uridine phosphorylase from Escherichia coli K-12 in hydrated reversed micelles of surfactant in organic solvent.

The catalytic activity of uridine phosphorylase from Escherichia coli K-12 entrapped in hydrated reversed micelles of aerosol OT (AOT) in octane has been studied as a function of the degree of hydration of micelles. It was shown that the catalytic activity reaches maximum values at ratios [H2O]/[AOT] equal to 8.4, 12.8, 16.1, and 18.6. On the basis of sedimentation data the conclusion has been made that the maximums of the catalytic activity of uridine phosphorylase correspond to monomeric, dimeric, trimeric, and tetrameric forms of the enzyme.

Bacterial Proteins↗
PubMed1998

Novel subtyping of intestinal metaplasia in the human stomach: brain-type glycogen phosphorylase expression in the proliferative zone and its relationship with carcinogenesis.

Although reports have suggested the incomplete type of intestinal metaplasia (IM) had a close correlation with carcinoma, considerable data showed no apparent relationship between the particular type of IM and the intestinal type carcinoma. The purpose of this study was to establish a novel classification of IM using brain-type glycogen phosphorylase (BGP) from a carcinogenetic viewpoint. The only isoform expressed in gastric cancer was BGP using polymerase chain reaction analysis. We studied 136 specimens with gastric carcinoma and the adjacent IM using specific anti-BGP antibody with its correlation to subtypes of IM, proliferating cell nuclear antigen-labeling index, and various oncogene products. Brain-type glycogen phosphorylase was expressed in 80.5% of the intestinal type and 18.8% of the diffuse type of carcinoma and in 87.5% and 41.6% in the generative zone of IM adjacent to cancer foci, respectively, whereas no reactivity was observed in the normal gastric mucosa. The proportion of the positivity in the cancer and IM was significantly greater in the intestinal-type carcinoma than in the diffuse type. The expression of BGP in the generative cells of IM had no significant correlation with the conventional type of IM. Intestinal metaplasias with BGP expression were significantly higher in a proliferating state than in those without BGP, and some of them that were coexpressed accumulated p53 in the generative cells. The relationship between IM with BGP in the generative cells and intestinal-type carcinoma was apparently closer than the conventional subtype of IM and gastric cancer. Intestinal-type carcinoma might arise from some of these proliferating cells with BGP.

Gastric Mucosa↗
PubMed1997

The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition.

The structure of a truncated form of the gamma-subunit of phosphorylase kinase (PHKgammat) has been solved in a ternary complex with a non-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti-parallel beta-sheet with the kinase activation segment, the region which in other kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide at a fixed distance from the gamma-phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate. The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognition of dimeric glycogen phosphorylase b is considered.

Adenylyl Imidodiphosphate↗
PubMed1993

McArdle's disease: a nonsense mutation in exon 1 of the muscle glycogen phosphorylase gene explains some but not all cases.

McArdle's disease is an inherited disease that results from a lack of functional muscle glycogen phosphorylase. We report here the identification of a C to T transition in exon 1 of the muscle phosphorylase gene found in all patients studied. This base pair mutation results in the substitution of a stop codon (TGA) for the codon (CGA) for Arg49 in the mature protein, and generates a novel restriction site for NlaIII. Of sixteen McArdle's patients, ten are homozygous for this mutation; the remainder are heterozygous. Additional unidentified mutations must lead to the McArdle's phenotype in the latter group of patients.

Base Sequence↗
PubMed1996

Mutation hotspots in the PHKA2 gene in X-linked liver glycogenosis due to phosphorylase kinase deficiency with atypical activity in blood cells (XLG2).

In five cases of X-linked liver glycogenosis subtype 2 (XLG2), we have identified mutations in the gene encoding the liver isoform of the phosphorylase kinase alpha subunit (PHKA2). XLG2 is a rare variant of X-linked phosphorylase kinase (Phk) deficiency of the liver. Whereas in the more common form of X-linked hepatic Phk deficiency, XLG1, the enzyme's activity is decreased both in liver and in blood cells, Phk activity in XLG2 is low in liver but normal or even enhanced in blood cells. Although missense, nonsense and splicesite mutations in the PHKA2 gene were recently identified in several cases of XLG1, no mutations have yet been described for XLG2 and a molecular explanation for the peculiar biochemical phenotype of XLG2 has been lacking. All mutations found in the present study result in non-conservative amino acid replacements of residues that are absolutely conserved between the alpha L, alpha M and beta subunits of Phk [H132P, H132Y, R186H (twice) and D299G]. Strikingly, in two pairs of cases the mutations affect the same codon. These results demonstrate that: (i) XLG2 is caused by mutations in PHKA2 and is therefore allelic with XLG1; and (ii) XLG2 mutations appear to cluster in limited sequence regions or even individual codons.

Amino Acid Sequence↗
PubMed1998

Identification of a mutation in liver glycogen phosphorylase in glycogen storage disease type VI.

Glycogen storage disease type VI (GSD6) defines a group of disorders that cause hepatomegaly and hypoglycemia with reduced liver phosphorylase activity. The course of these disorders is generally mild, but definitive diagnosis requires invasive procedures. We analyzed a Mennonite kindred with an autosomal recessive form of GSD6 to determine the molecular defect and develop a non-invasive diagnostic test. Linkage analysis was performed using genetic markers flanking the liver glycogen phosphorylase gene ( PYGL ), which was suspected to be the cause of the disorder on biochemical grounds. Mennonite GSD6 was linked to the PYGL locus with a multipoint LOD score of 4.7. The PYGL gene was analyzed for mutations by sequencing genomic DNA. Sequencing of genomic DNA revealed a splice site abnormality of the intron 13 splice donor. Confirmation of the genomic mutation was performed by sequencing RT-PCR products, which showed heterogeneous PYGL mRNA lacking all or part of exon 13 in affected persons. This study is the first to demonstrate that a mutation in the PYGL gene can cause GSD6. This mutation is estimated to be present on 3% of Mennonite chromosomes and the disease affects 0.1% of that population. Determination of this mutation provides a basis for the development of a simple and non-invasive diagnostic test for the disease and the carrier state in this population and confirms biochemical data showing the importance of this gene in glucose homeostasis.

Base Sequence↗
PubMed2003

Mutagenesis of the dimer interface region of Corynebacterium callunae starch phosphorylase perturbs the phosphate-dependent conformational relay that enhances oligomeric stability of the enzyme.

We have used alanine-scanning site-directed mutagenesis of the dimer contact region of starch phosphorylase from Corynebacterium callunae to explore the relationship between a protein conformational change induced by phosphate binding and the up to 500-fold kinetic stabilization of the functional quarternary structure of this enzyme when phosphate is present. Purified mutants (at positions Ser-224, Arg-226, Arg-234, and Arg-242) were characterized by Fourier transform-infrared (FT-IR) spectroscopy and enzyme activity measurements at room temperature and under conditions of thermal denaturation. Difference FT-IR spectra of wild type and mutants in (2)H(2)O solvent revealed small changes in residual amide II band intensities at approximately 1,550 cm(-1), indicating that (1)H/(2)H exchange in the wild type is clearly perturbed by the mutations. Decreased (1)H/(2)H exchange in comparison to wild type suggests formation of a more compact protein structure in S224A, R234A, and R242A mutants and correlates with rates of irreversible thermal denaturation at 45 degrees C that are up to 10-fold smaller for the three mutants than the wild type. By contrast, the mutant R226A inactivates 2.5-fold faster at 45 degrees C and shows a higher (1)H/(2)H exchange than the wild type. Phosphate (20 mM) causes a greater change in FT-IR spectra of the wild type than in those of S224A and 234A mutants and leads to a 5-fold higher stabilization of the wild type than the two mutants. Therefore, structural effects of phosphate binding leading to kinetic stability of wild-type starch phosphorylase are partially complemented in the S224A and R234A mutants. Infrared spectroscopic measurements were used to compare thermal denaturations of the mutants and the wild type in the absence and presence of stabilizing oxyanion. The broad denaturation transition of unliganded wild type in the range 40-50 degrees C is reduced in the S224A and R234A mutants, and this reflects mainly a shift of the onset of denaturation to a 4-5 degrees C higher value.

Alanine↗