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1.
Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer consisting of four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha, beta, and delta, the last being endogenous calmodulin). The enzyme is activated by a variety of effectors acting through its regulatory subunits. To probe the quaternary structure of nonactivated and activated forms of the kinase, we used the heterobifunctional, photoreactive cross-linker N-5-azido-2-nitrobenzoyloxysuccinimide. Mono-derivatization of the holoenzyme with the succinimidyl group, followed by photoactivation of the covalently attached azido group, resulted in intramolecular cross-linking to form two distinct heterodimers: a major (alphagamma) and a minor (betadelta) conjugate. Formation of both conjugates was significantly altered in activated conformations of the enzyme induced by phosphorylation, alkaline pH, and several allosteric activators (ADP, exogenous calmodulin/Ca2+, and Ca2+ alone). Of these activating mechanisms, all increased formation of alphagamma, except Ca2+ alone, which inhibited its formation. When cross-linking was carried out at alkaline pH or in the presence of ADP or exogenous calmodulin/Ca2+, the cross-linked enzyme remained activated following removal of the activators; however, cross-linking in the presence of Ca2+ resulted in sustained inhibition. The results indicate that perturbations in the subunit cross-linking forming the alphagamma dimer reflect the subsequent extent of sustained activation of the holoenzyme that is measured. The region cross-linked to the catalytic gamma subunit was confined to the C-terminal 1/6th of the alpha subunit, which contains known regulatory regions. These results suggest that activators of the phosphorylase kinase holoenzyme perturb interactions between the C-terminal region of the inhibitory alpha subunit and the catalytic gamma subunit, ultimately leading to activation of the latter. 相似文献
2.
The catalytic subunit of phosphorylase b kinase (gamma) and an engineered truncated form (gamma-trc, residues 1-297) have been expressed in Escherichia coli. The truncated protein included the entire catalytic domain as defined by sequence alignment with other protein kinases but lacked the putative calmodulin binding domain. Full-length protein was produced in insoluble aggregates. Some activity was regenerated by solubilization in urea and dilution into renaturating buffer but the activity was found to be associated with a smaller molecular weight component. Full-length protein could not be refolded successfully. The truncated gamma subunit was produced in the soluble fraction of the cell as well as in inclusion bodies. The insoluble protein was refolded by dilution from urea and purified to homogeneity, in a one step separation on DEAE-Sepharose to give a protein mol. wt 32,000 +/- 2000 with a high sp. act. of 5.3 mumol 32P incorporated into phosphorylase b(PPB)/min/nmol. Kinetic parameters gave Km for ATP 46 +/- 3 microM and Km for PPb 27 +/- 1 microM. The sp. act. and the Km values are comparable to those observed for the activated holoenzyme and indicate that the gamma-trc retains the substrate recognition and catalytic properties. The ratio of activities at pH 6.8/8.2 was 0.84. gamma-trc was inhibited by ADP with a Ki of 52 microM and was sensitive to activation by Mg2+ and inhibition by Mn2+, properties that are characteristic of the holoenzyme and the isolated gamma subunit. Calmodulin which confers calcium sensitivity on the isolated gamma subunit had no effect on the enzymic properties of gamma-trc.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
《Genetic analysis, techniques and applications》1993,10(3-4):99-101
The catalytic subunit, γ, of phosphorylase kinase contains two calmodulin-binding sequences that define a domain in γ that is homologous to the troponin-C-binding domain in troponin I. The homology is based on both sequence and functional similarities. To account for this homology, it has been proposed that the calmodulin-binding sequences in γ and the troponin-C-binding domain in troponin I have evolved from a common ancestor. We investigated this possibility by comparing the exon structure of the γ gene with that of the troponin-I gene over their homologous domains. In the quail troponin-I gene, it is known that the entire troponin-C-binding domain is encoded by a single exon. However, two exons are found to encode the calmodulin-binding domain in the γ gene from mouse. This result indicates that convergent evolution may be responsible for the sequence and functional similarities between the homologous domains in troponin I and γ. 相似文献
4.
The phosphorylase kinase holoenzyme from skeletal muscle is composed of a catalytic and three different regulatory subunits. Analysis of the kinetic mechanism of the holoenzyme is complicated because both the natural substrate phosphorylase b and also phosphorylase kinase itself have allosteric binding sites for adenine nucleotides. In the case of the kinase, these allosteric sites are not on the catalytic subunit. We have investigated the kinetic mechanism of phosphorylase kinase by using its isolated catalytic gamma-subunit (activated by calmodulin) and an alternative peptide substrate (SDQEKRKQISVRGL) corresponding to the convertible region of phosphorylase b, thus eliminating from our system all known allosteric binding sites for nucleotides. This peptide has been previously employed to study the kinetic mechanism of the kinase holoenzyme before the existence of the allosteric sites on the regulatory subunits was suspected [Tabatabai, L. B., & Graves, D. J. (1978) J. Biol. Chem. 253, 2196-2202]. This peptide was determined to be as good an alternative substrate for the isolated catalytic subunit as it was for the holoenzyme. Initial velocity data indicated a sequential kinetic mechanism with apparent Km's for MgATP and peptide of 0.07 and 0.47 mM, respectively. MgADP used as product inhibitor showed competitive inhibition against MgATP and noncompetitive inhibition against peptide, whereas with phosphopeptide as product inhibitor, the inhibition was competitive against both MgATP and peptide. The initial velocity and product inhibition studies were consistent with a rapid equilibrium random mechanism with one abortive complex, enzyme-MgADP-peptide. The substrate-directed, dead-end inhibitors 5'-adenylyl imidodiphosphate and Asp-peptide, in which the convertible Ser of the alternative peptide substrate was replaced with Asp, were competitive inhibitors toward their like substrates and noncompetitive inhibitors toward their unlike substrates, further supporting a random mechanism, which was also the conclusion from the report cited above that used the holoenzyme. 相似文献
5.
Inhibition of the catalytic subunit of phosphorylase kinase by its alpha/beta subunits 总被引:3,自引:0,他引:3
The subunits of phosphorylase kinase are separated and isolated in high yield by gel filtration chromatography in pH 3.3 phosphate buffer containing 8 M urea. Three protein peaks are obtained: the alpha and beta subunits coelute in the first, whereas the gamma and delta subunits are separate peaks. Upon dilution of the denaturant, catalytic activity reappears, associated only with the gamma subunit. As has been previously observed (Kee, S.M., and Graves, D.J. (1986) J. Biol. Chem. 261, 4732-4737), addition of calmodulin dramatically stimulates the reactivation of gamma. Inclusion of increasing amounts of the alpha/beta subunit mixture in the renaturation progressively decreases the activity of the renatured gamma or gamma-calmodulin. This inhibition by alpha/beta is likely due to specific interactions with the gamma subunit because the inhibition is less at pH 8.2 than at pH 6.8 and less when equivalent amounts of phosphorylated alpha/beta subunits are used (both alkaline pH and phosphorylation are known to stimulate the activity of the holoenzyme). These results suggest that the role of either the alpha or beta subunits, or perhaps both, in the nonactivated (alpha 2 beta 2 gamma 2 delta 2)2 complex of phosphorylase kinase is to suppress the activity of the gamma subunit and that activation of the enzyme, by phosphorylation for instance, is due to deinhibition caused by release of this quaternary constraint by alpha and/or beta upon gamma. 相似文献
6.
Purification and characterization of catalytic fragments of phosphorylase kinase gamma subunit missing a calmodulin-binding domain 总被引:1,自引:0,他引:1
W R Harris D A Malencik C M Johnson S A Carr G D Roberts C A Byles S R Anderson L M Heilmeyer E H Fischer J W Crabb 《The Journal of biological chemistry》1990,265(20):11740-11745
A catalytic fragment preparation of rabbit muscle phosphorylase kinase produced by limited chymotryptic digestion was isolated and identified as the NH2-terminal region of the gamma subunit by Edman degradation. Mass spectral analysis, gas phase sequence analysis, and amino acid analysis of the active fragment carboxyl-terminal peptides revealed multiple COOH termini generated at residues Tyr290, Arg296, and Phe298 in the gamma subunit sequence. These active fragment species are about 24% smaller than the gamma subunit (Mr 44,673) and range in size from Mr 33,279 to Mr 34,275. The active fragment preparation exhibits a specific activity about 6-fold higher than that of the gamma subunit-calmodulin complex. Calmodulin confers calcium sensitivity to the gamma subunit but has no effect on the enzymatic properties of active fragment. Affinity measurements demonstrated a dissociation constant of 0.7 microM for active fragment binding to dansylcalmodulin, a value about 28-fold weaker than reported for the gamma subunit. These data support the presence of a calmodulin binding domain in the COOH-terminal region of the gamma subunit. 相似文献
7.
The interaction between calmodulin (CaM) and two synthetic peptides, C20W and C24W, corresponding to parts of the calmodulin-binding domain of the Ca2+ pump of human erythrocytes, has been studied by using small-angle X-ray scattering (SAXS). The total length of the CaM-binding domain of the enzyme is estimated to be 28 amino acids. C20W contains the 20 N-terminal amino acids of this domain, C24W the 24 C-terminal amino acids. The experiments have shown that the binding of either peptide results in a complex with a radius of gyration (Rg) smaller than that of CaM. The complex between CaM and C20W revealed an interatomic length distribution function, P(r), similar to that of calmodulin alone, indicating that the complex retains an extended, dumbbell-shaped structure. By contrast, the binding of C24W resulted in the formation of a globular structure similar to those observed with many other CaM-binding peptides. 相似文献
8.
The CNBr peptides, CBPa and CBPb, corresponding to the N-terminal 1-91 amino acid residues of glycogen-phosphorylase a and b, respectively, were purified and characterized. CD, 31P-NMR and fluorescence spectroscopy were used to assess the structural organization of the cyanogen bromide peptides in solution. The cyanogen bromide peptides yielded 21% of alpha-helical structures by CD compared to a calculated value of 36.3%. These peptides interact with calmodulin which induces measurable alpha-helices in the cyanogen bromide peptides. The helix stabilizing reagent, trifluoroethanol, induces high numbers of alpha-helices in CBP, thereby demonstrating the conformation fluidity of this peptide. The dissociation constants for calmodulin and CBP estimated by fluorescence titrations were 36.0 and 29.9 nM for CBPb in the presence of Ca2+ and EGTA, respectively. The phosphorylated residue in CBPa causes a decrease in binding interactions with calmodulin and corresponding values obtained for CBPa by fluorescence titration are 56.0 and 141.0 nM, respectively. The Ser-P-14 of CBPa is titratable, yielding a pKa = 5.45 and a Hill coefficient of 1.5. A helical wheel analysis using a computer program in PC/GENE of the CBP shows that peptide stretches in the alpha-1 and alpha-2 helices are most basic and fairly amphiphilic and therefore represent the most probable segment for CaM binding. It is this structural character of these segments which presumably confer the ability to bind CaM and facilitate some of the allosteric transitions of glycogen phosphorylase. 相似文献
9.
The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, , of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN"), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any constructs; however, PhbN" interacted with a region of (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN" was also screened for interactions against a variety of fragments of the catalytic subunit of PhK; however, no interactions were detected, even with fulllength . Our results support the idea that amino acid residues proximal to the convertible serine of Phb are important for its specific interaction with the catalytic subunit of PhK, but that regions distinct from the convertible serine residue of Phb and from the catalytic domain of PhK may also be involved in the interaction of these two proteins. 相似文献
10.
Phosphorylase kinase contains four approximately equivalent binding sites for 1-anilinonaphthalene-8-sulfonate (1,8-ANS). Measurements of the time decay of fluorescence anisotropy have failed to give any indication of internal degrees of rotational freedom involving a significant portion of the tertiary structure. In the presence of 1 mM Ca2+, calmodulin binds one molecule of 1,8-ANS. No binding occurs in the absence of Ca2+. The binding is strongly temperature-dependent, a decrease in binding occurring with increasing temperature. Determinations of the time decay of fluorescence anisotropy indicate the presence of internal rotations, which become more important with increasing temperature. Complex formation between phosphorylase kinase and calmodulin reduces the binding of 1,8-ANS. 相似文献
11.
Boulatnikov IG Nadeau OW Daniels PJ Sage JM Jeyasingham MD Villar MT Artigues A Carlson GM 《Biochemistry》2008,47(27):7228-7236
Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca (2+)-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 approximately 0.45 microM) PhK's conversion of GP b. When a short synthetic peptide substrate was substituted for GP b, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the gamma subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory beta subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the beta subunit, specifically an alphagammadelta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GP b. Moreover, GAPDH had no effect on the conversion of GP b by the alphagammadelta subcomplex. The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle. 相似文献
12.
Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca(2+) ions, of the (alphabetagammadelta)(4) phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory alpha and catalytic gamma subunits. The gamma subunit is also known to interact with the delta subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca(2+) ions. In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits. The yeast two-hybrid system indicated that regions near the C termini of the gamma (residues 343-386) and alpha (residues 1060-1237) subunits interact. The association of this region of alpha with gamma was corroborated by the isolation of a cross-linked fragment of alpha containing residues 1015-1237 from an alpha-gamma dimer that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker. Because the region of gamma that we found to interact with alpha has previously been shown to contain a high affinity binding site for calmodulin (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163), we tested the influence of Ca(2+) on the conformation of the alpha subunit and found that the region of alpha that interacts with gamma was, in fact, perturbed by Ca(2+). The results herein support the existence of a Ca(2+)-sensitive communication network among the delta, gamma, and alpha subunits, with the regulatory domain of gamma being the primary mediator. The similarity of such a Ca(2+)-dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the gamma subunit of PhK. 相似文献
13.
Hilde Damaschun Gregor Damaschun Dietrich Zirwer Rolf Misselwitz Irina A. Zalenskaya Vladimir I. Vorobev 《International journal of biological macromolecules》1983,5(4):250-252
The nucleosome core histone complex in solution at 2 M NaCl and pH 7 has a radius of gyration Rs, of 3.48 nm and a maximum dimension, L, of 12 nm. Its shape is disc-like with a mean thickness of 3 nm. The radius of gyration determined by us is of the same value as the radius of gyration of the complex in intact core particles (Braddock) et al., Biopolymers 1981, 20, 327). Thus, we conclude that the basic histone tails of the protein complex project about 2 nm from its central part. 相似文献
14.
The strength of the interaction between the catalytic and regulatory subunits in protein kinase A differs among species. The linker region from regulatory subunits is non-conserved. To evaluate the participation of this region in the interaction with the catalytic subunit, we have assayed its effect on the enzymatic properties of the catalytic subunit. Protein kinase A from three fungi, Mucor rouxii, Mucor circinelloides and Saccharomyces cerevisiae have been chosen as models. The R-C interaction is explored by using synthetic peptides of 8, 18 and 47 amino acids, corresponding to the R subunit autophosphorylation site plus a variable region toward the N terminus (0, 10, or 39 residues). The Km of the catalytic subunits decreased with the length of the peptide, while the Vmax increased. Viscosity studies identified product release as the rate limiting step for phosphorylation of the longer peptides. Pseudosubstrate derivatives of the 18 residue peptides did not display a competitive inhibition behavior toward either kemptide or a bona fide protein substrate since, at low relative pseudosubstrate/substrate concentration, stimulation of kemptide or protein substrate phosphorylation was observed. The behavior was mimicked by intact R. We conclude that in addition to its negative regulatory role, the R subunit stimulates C activity via distal interactions. 相似文献
15.
Mutations in the liver isoform of the Phosphorylase Kinase (PhK) alpha subunit (PHKA2 gene) cause X-linked liver glycogenosis (XLG), the most frequent type of PhK deficiency (glycogen-storage disease type IX). XLG patients can be divided in two subgroups, with similar clinical features but different activity of PhK (decreased in liver and blood cells for XLG-I and low in liver but normal or enhanced in blood cells for XLG-II). Here, we show that the PHKA2 missense mutations and small in-frame deletions/insertions are concentrated into two domains of the protein, which were recently described. In the N-terminal glucoamylase domain, mutations (principally leading to XLG-II) are clustered within the predicted glycoside-binding site, suggesting that they may have a direct impact on a possible hydrolytic activity of the PhK alpha subunit, which remains to be demonstrated. In the C-terminal calcineurin B-like domain (domain D), mutations (principally leading to XLG-I) are clustered in a region predicted to interact with the regulatory region of the PhK catalytic subunit and in a region covering this interaction site. Altogether, these results show that PHKA2 missense mutations or small in-frame deletions/insertions may have a direct impact on the PhK alpha functions and provide a framework for further experimental investigation. 相似文献
16.
Ablooglu AJ Frankel M Rusinova E Ross JB Kohanski RA 《The Journal of biological chemistry》2001,276(50):46933-46940
Low catalytic efficiency of protein kinases often results from intrasteric inhibition caused by the activation loop blocking the active site. In the insulin receptor's kinase domain, Asp-1161 and Tyr-1162 in the peptide substrate-like sequence of the unphosphorylated activation loop can interact with four invariant residues in the active site: Lys-1085, Asp-1132, Arg-1136, and Gln-1208. Contributions of these six residues to intrasteric inhibition were tested by mutagenesis, and the unphosphorylated kinase domains were characterized. The mutations Q1208S, K1085N, and Y1162F each relieved intrasteric inhibition, increasing catalytic efficiency but without changing the rate-limiting step of the reaction. The mutants R1136Q and D1132N were virtually inactive. Steric accessibility of the active site was ranked by relative changes in iodide quenching of intrinsic fluorescence, and A-loop conformation was ranked by limited tryptic cleavage. Together these ranked the openness of the active site cleft as R1136Q approximately D1132N > or = D1161A > Y1162F approximately K1085N > Q1208S > or = wild-type. These findings demonstrate the importance of specific invariant residues for intrasteric inhibition and show that diverse activation loop conformations can produce similar steady-state kinetic properties. This suggests a broader range of regulatory properties for the activation loop than expected from a simple off-versus-on switch for kinase activation. 相似文献
17.
18.
Thymidine phosphorylase (TP) is a dual substrate enzyme with two domains. Each domain binds a substrate. In the crystal structure of Escherichia coli TP, the two domains are arranged so that the two substrate binding sites are too far away for the two substrates to directly react. Molecular dynamics simulations reveal a different structure of the enzyme in which the two domains have moved to place the two substrates in close contact. This structure has a root-mean-square deviation from the crystal structure of 4.1 A. Quantum mechanical calculations using this structure find that the reaction can proceed by a direct nucleophilic attack with a low barrier. This mechanism is not feasible in the crystal structure environment and is consistent with the mechanism observed for other N-glycosidic enzymes. Important catalytic roles are found for the three highly conserved residues His 85, Arg 171, and Lys 190. 相似文献
19.
We have studied the interactions between calmodulin (CaM) and three target peptides from the death-associated protein kinase (DAPK) protein family using both experimental and modeling methods, aimed at determining the details of the underlying biological regulation mechanisms. Experimentally, calorimetric binding free energies were determined for the complexes of CaM with peptides representing the DAPK2 wild-type and S308D mutant, as well as DAPK1. The observed affinity of CaM was very similar for all three studied peptides. The DAPK2 and DAPK1 peptides differ significantly in sequence and total charge, while the DAPK2 S308D mutant is designed to model the effects of DAPK2 Ser308 phosphorylation. The crystal structure of the CaM-DAPK2 S308D mutant peptide is also reported. The structures of CaM-DAPK peptide complexes present a mode of CaM-kinase interaction, in which bulky hydrophobic residues at positions 10 and 14 are both bound to the same hydrophobic cleft. To explain the microscopic effects underlying these interactions, we performed free energy calculations based on the approximate MM-PBSA approach. For these highly charged systems, standard MM-PBSA calculations did not yield satisfactory results. We proposed a rational modification of the approach which led to reasonable predictions of binding free energies. All three complexes are strongly stabilized by two effects: electrostatic interactions and buried surface area. The strong favorable interactions are to a large part compensated by unfavorable entropic terms, in which vibrational entropy is the largest contributor. The electrostatic component of the binding free energy followed the trend of the overall peptide charge, with strongest interactions for DAPK1 and weakest for the DAPK2 mutant. The electrostatics was dominated by interactions of the positively charged residues of the peptide with the negatively charged residues of CaM. The nonpolar binding free energy was comparable for all three peptides, the largest contribution coming from the Trp305. About two-thirds of the buried surface area corresponds to nonpolar residues, showing that hydrophobic interactions play an important role in these CaM-peptide complexes. The simulation results agree with the experimental data in predicting a small effect of the S308D mutation on CaM interactions with DAPK2, suggesting that this mutation is not a good model for the S308 phosphorylation. 相似文献
20.
Acetohydroxyacid synthase (EC 4.1.3.18; AHAS) catalyzes the initial step in the formation of the branched-chain amino acids. The enzyme from most bacteria is composed of a catalytic subunit, and a smaller regulatory subunit that is required for full activity and for sensitivity to feedback regulation by valine. A similar arrangement was demonstrated recently for yeast AHAS, and a putative regulatory subunit of tobacco AHAS has also been reported. In this latter case, the enzyme reconstituted from its purified subunits remained insensitive to feedback inhibition, unlike the enzyme extracted from native plant sources. Here we have cloned, expressed in Escherichia coli, and purified the AHAS regulatory subunit of Arabidopsis thaliana. Combining the protein with the purified A. thaliana catalytic subunit results in an activity stimulation that is sensitive to inhibition by valine, leucine, and isoleucine. Moreover, there is a strong synergy between the effects of leucine and valine, which closely mimics the properties of the native enzyme. The regulatory subunit contains a sequence repeat of approximately 180 residues, and we suggest that one repeat binds leucine while the second binds valine or isoleucine. This proposal is supported by reconstitution studies of the individual repeats, which were also cloned, expressed, and purified. The structure and properties of the regulatory subunit are reminiscent of the regulatory domain of threonine deaminase (EC 4.2.1.16), and it is suggested that the two proteins are evolutionarily related. 相似文献