Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsin-like enzymes and on the increase in catalytic efficiency.     

Phosphorylation and mutations of Ser(16) in human phenylalanine hydroxylase. Kinetic and structural effects

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser(16) by cyclic AMP-dependent protein kinase is a post-translational modification that increases its basal activity and facilitates its activation by the substrate l-Phe. So far there is no structural information on the flexible N-terminal tail (residues 1-18), including the phosphorylation site. To get further insight into the molecular basis for the effects of phosphorylation on the catalytic efficiency and enzyme stability, molecular modeling was performed using the crystal structure of the recombinant rat enzyme. The most probable conformation and orientation of the N-terminal tail thus obtained indicates that phosphorylation of Ser(16) induces a local conformational change as a result of an electrostatic interaction between the phosphate group and Arg(13) as well as a repulsion by Glu(280) in the loop at the entrance of the active site crevice structure. The modeled reorientation of the N-terminal tail residues (Met(1)-Leu(15)) on phosphorylation is in agreement with the observed conformational change and increased accessibility of the substrate to the active site, as indicated by circular dichroism spectroscopy and the enzyme kinetic data for the full-length phosphorylated and nonphosphorylated human PAH. To further validate the model we have prepared and characterized mutants substituting Ser(16) with a negatively charged residue and found that S16E largely mimics the effects of phosphorylation of human PAH. Both the phosphorylated enzyme and the mutants with acidic side chains instead of Ser(16) revealed an increased resistance toward limited tryptic proteolysis and, as indicated by circular dichroism spectroscopy, an increased content of alpha-helical structure. In agreement with the modeled structure, the formation of an Arg(13) to Ser(16) phosphate salt bridge and the conformational change of the N-terminal tail also explain the higher stability toward limited tryptic proteolysis of the phosphorylated enzyme. The results obtained with the mutant R13A and E381A further support the model proposed for the molecular mechanism for the activation of the enzyme by phosphorylation.     

Phosphorylation-induced conformational changes in a mitogen-activated protein kinase substrate. Implications for tyrosine hydroxylase activation

Mitogen-activated protein (MAP) kinase-mediated phosphorylation of specific residues in tyrosine hydroxylase leads to an increase in enzyme activity. However, the mechanism whereby phosphorylation affects enzyme turnover is not well understood. We used a combination of fluorescence resonance energy transfer (FRET) measurements and molecular dynamics simulations to explore the conformational free energy landscape of a 10-residue MAP kinase substrate found near the N terminus of the enzyme. This region is believed to be part of an autoregulatory sequence that overlies the active site of the enzyme. FRET was used to measure the effect of phosphorylation on the ensemble of peptide conformations, and molecular dynamics simulations generated free energy profiles for both the unphosphorylated and phosphorylated peptides. We demonstrate how FRET transfer efficiencies can be calculated from molecular dynamics simulations. For both the unphosphorylated and phosphorylated peptides, the calculated FRET efficiencies are in excellent agreement with the experimentally determined values. Moreover, the FRET measurements and molecular simulations suggest that phosphorylation causes the peptide backbone to change direction and fold into a compact structure relative to the unphosphorylated state. These results are consistent with a model of enzyme activation where phosphorylation of the MAP kinase substrate causes the N-terminal region to adopt a compact structure away from the active site. The methods we employ provide a general framework for analyzing the accessible conformational states of peptides and small molecules. Therefore, they are expected to be applicable to a variety of different systems.     

The AP-1 repressor, JDP2, is a bona fide substrate for the c-Jun N-terminal kinase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine and inhibited by its cofactor tetrahydrobiopterin (BH(4)). The crystal structure of PAH revealed that the N-terminal sequence of the enzyme (residues 19-29) partially covered the enzyme active site, and suggested its involvement in regulation. We show that the protein lacking this N-terminal sequence does not require activation by phenylalanine, shows an altered structural response to phenylalanine, and is not inhibited by BH(4). Our data support the model where the N-terminal sequence of PAH acts as an intrasteric autoregulatory sequence, responsible for transmitting the effect of phenylalanine activation to the active site.     

Structural basis of autoregulation of phenylalanine hydroxylase.

Phenylalanine hydroxylase converts phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. It is tightly regulated by the substrates phenylalanine and tetrahydrobiopterin and by phosphorylation. We present the crystal structures of dephosphorylated and phosphorylated forms of a dimeric enzyme with catalytic and regulatory properties of the wild-type protein. The structures reveal a catalytic domain flexibly linked to a regulatory domain. The latter consists of an N-terminal autoregulatory sequence (containing Ser 16, which is the site of phosphorylation) that extends over the active site pocket, and an alpha-beta sandwich core that is, unexpectedly, structurally related to both pterin dehydratase and the regulatory domains of metabolic enzymes. Phosphorylation has no major structural effects in the absence of phenylalanine, suggesting that phenylalanine and phosphorylation act in concert to activate the enzyme through a combination of intrasteric and possibly allosteric mechanisms.     

Missense mutations in the N-terminal domain of human phenylalanine hydroxylase interfere with binding of regulatory phenylalanine

Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene. Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective. Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46-48 (GAL) and 65-69 (IESRP), two motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V, T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2-120) of human PAH were expressed as fusion proteins in Escherichia coli. Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency.     

Role of the N-terminal region of the regulatory light chain in the dephosphorylation of myosin by myosin light chain phosphatase.

Myosin regulatory light chain (RLC) is phosphorylated at various sites at its N-terminal region, and heterotrimeric myosin light chain phosphatase (MLCP) has been assigned as a physiological phosphatase that dephosphorylates myosin in vivo. Specificity of MLCP toward the various phosphorylation sites of RLC was studied, as well as the role of the N-terminal region of RLC in the dephosphorylation of myosin by MLCP. MLCP dephosphorylated phosphoserine 19, phosphothreonine 18, and phosphothreonine 9 efficiently with almost identical rates, whereas it failed to dephosphorylate phosphorylated serine 1/serine 2. Deletion of the N-terminal seven amino acid residues of RLC markedly decreased the dephosphorylation rate of phosphoserine 19 of RLC incorporated in the myosin molecule, whereas this deletion did not significantly affect the dephosphorylation rate of isolated RLC. On the other hand, deletion of only four N-terminal amino acid residues showed no effect on dephosphorylation of phosphoserine 19 of incorporated RLC. The inhibition of dephosphorylation by deletion of the seven N-terminal residues was also found with the catalytic subunit of MLCP. Phosphorylation at serine 1/serine 2 and threonine 9 did not influence the dephosphorylation rate of serine 19 and threonine 18 by MLCP. These results suggest that the N-terminal region of RLC plays an important role in substrate recognition of MLCP.     

Phosphorylation of the N-terminal portion of tyrosine hydroxylase triggers proteasomal digestion of the enzyme

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its N-terminus plays a critical role in the intracellular stability of the enzyme. In the present study, we investigated the mechanism by which the N-terminal region of TH affects this stability. TH molecules phosphorylated at their Ser31 and Ser40 were localized predominantly in the cytoplasm of PC12D cells. However, those molecules phosphorylated at Ser19 were found mainly in the nucleus, whereas they seemed to be negligible in the cytoplasm. The inhibition of proteasomes increased the quantity of TH molecules phosphorylated at their Ser19 and Ser40, although it did not increase that of TH molecules or that of TH phosphorylated at its Ser31. The inhibition of autophagy did not affect the amount of the TH molecule or that of its three phosphorylated forms. Deletion mutants of human TH type-1 lacking the N-terminal region containing the three phosphorylation sites possessed high stability of the enzyme in PC12D cells. These results suggest that the phosphorylation of the N-terminal portion of TH regulates the degradation of this enzyme by the ubiquitin–proteasome pathway.     

Characterization of mitogen-activated protein kinase (MAPK) dimers

Phosphorylated ERK2 has an increased capacity to form homodimers relative to unphosphorylated ERK2. We have characterized the nature of the ERK2 dimer and have mutated residues in the crystal dimer interface to examine the impact of dimerization on ERK2 activity. Analysis of the mutants by gel filtration indicates that at least five residues must be mutated simultaneously to produce an ERK2 mutant that is predominantly monomeric. Mutants, whether monomers or dimers, have specific protein kinase activities under fixed assay conditions that are roughly equivalent to wild-type ERK2. The ratio of dimers to monomers is increased as the salt concentration increases, consistent with a strong hydrophobic contribution to the energy of dimer formation. ERK2 dimerization also requires divalent cations. Sedimentation analysis indicates that the related c-Jun N-terminal kinase SAPKalphaI/JNK2 also forms dimers, but dimerization displays no dependence on phosphorylation; the unphosphorylated and phosphorylated forms of the kinase behave similarly, with low micromolar dimer dissociation constants.     

Amino acid sequence of in vivo phosphorylation sites in the main intrinsic protein (MIP) of lens membranes

The main intrinsic membrane protein of the lens fiber cell, MIP, has been previously shown to be phosphorylated in preparations of lens fragments. Phosphorylation occurred on serine residues near the cytoplasmic C-terminus of the molecule. Since MIP is thought to function as a channel protein in lens plasma membranes, possibly as a cell-to-cell channel protein, phosphorylation could regulate the assembly or gating of these channels. We sought to identify the specific serines which are phosphorylated in order to help identify the kinases involved in regulating MIP function. To this end we purified a peptide fragment from native membranes that had not been subjected to any exogenous kinases or kinase activators. Any phosphorylation detected in these fragments must be due to cellular phosphorylation and thus is termed in vivo phosphorylation. Purified membranes were also phosphorylated with cAMP-dependent protein kinase to determine the mobility of phosphorylated and unphosphorylated MIP-derived peptides on different HPLC columns and to determine possible cAMP-dependent protein kinase phosphorylation sites. Lens membranes, which contain 50-60% of the protein as MIP, were digested with lysylendopeptidase C. Peptides were released from the C-terminal region of MIP and a major product of 21-22 kDa remained membrane-associated. Separation of the lysylendopeptidase-C-released peptides on C8 reversed-phase HPLC demonstrated that one of these fragments, corresponding to residues 239-259 in MIP, was partially phosphorylated. The phosphorylated and nonphosphorylated forms of this peptide were separated on QAE HPLC. In vivo phosphorylation sites were found at residues 243 and 245 through phosphoserine modification via ethanethiol and sequence analysis. Phosphorylation was never detected on serine 240. The phosphorylation level of serine 243 could be increased by incubation of membranes with cAMP-dependent protein kinase under standard assay conditions. Other kinases that phosphorylate serines found near acidic amino acids must be responsible for the in vivo phosphorylation demonstrated at serine 245.     

Structural and biochemical evidence for an autoinhibitory role for tyrosine 984 in the juxtamembrane region of the insulin receptor

Tyrosine 984 in the juxtamembrane region of the insulin receptor, between the transmembrane helix and the cytoplasmic tyrosine kinase domain, is conserved among all insulin receptor-like proteins from hydra to humans. Crystallographic studies of the tyrosine kinase domain and proximal juxtamembrane region reveal that Tyr-984 interacts with several other conserved residues in the N-terminal lobe of the kinase domain, stabilizing a catalytically nonproductive position of alpha-helix C. Steady-state kinetics measurements on the soluble kinase domain demonstrate that replacement of Tyr-984 with phenylalanine results in a 4-fold increase in kcat in the unphosphorylated (basal state) enzyme. Moreover, mutation of Tyr-984 in the full-length insulin receptor results in significantly elevated receptor phosphorylation levels in cells, both in the absence of insulin and following insulin stimulation. These data demonstrate that Tyr-984 plays an important structural role in maintaining the quiescent, basal state of the insulin receptor. In addition, the structural studies suggest a possible target site for small molecule activators of the insulin receptor, with potential use in the treatment of noninsulin-dependent diabetes mellitus.     

Amino acid sequence of the amphiphilic phosphocarrier protein factor IIILac of the lactose-specific phosphotransferase system of Staphylococcus

The lactose-specific factor III of the phosphotransferase system of Staphylococcus aureus is an amphiphilic trimeric protein composed of identical subunits. It is hydrophilic in its unphosphorylated state and can be isolated from the cytoplasmic protein fraction. It becomes a constituent of the membrane-bound phosphotransferase complex upon phosphorylation of a single histidyl residue. The sequence of S. aureus factor IIILac was determined and revealed that the subunits consist of 103 residues corresponding to a Mr of 11 367 and of 34 101 for the native trimer: (sequence; see text) According to this sequence and previous work histidine residue 82 located in the C-terminal part of the polypeptide chain is phosphorylated at the N-3 position by phosphoenolpyruvate, enzyme I, and histidine-containing phosphocarrier protein. The N-terminal part of the protein comprising approximately one-third of the chain exhibits in vitro affinity toward membrane-bound enzyme IILac.     

Fructose-1,6-bisphosphatase from rat liver. A comparison of the kinetics of the unphosphorylated enzyme and the enzyme phosphorylated by cyclic AMP-dependent protein kinase

A purification procedure for rat hepatic fructose-1,6-bisphosphatase, described earlier, has been improved, resulting in an enzyme preparation with a neutral pH optimum and with both phosphorylatable serine residues present. The subunit Mr was 40,000. Phosphorylation in vitro with cyclic AMP-dependent protein kinase resulted in the incorporation of 1.4 mol of phosphate/mol of subunit and led to an almost 2-fold decrease in apparent Km for fructose-1,6-bisphosphate. In contrast to yeast fructose-1,6-bisphosphatase, fructose-2,6-bisphosphate had no effect on the rate of phosphorylation or dephosphorylation of the intact enzyme. The effects of the composition of the assay medium, with regard to buffering substance and Mg2+ concentration, on the apparent Km values of phosphorylated and unphosphorylated enzyme were investigated. The kinetics of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase were studied with special reference to the inhibitory effects of adenine nucleotides and fructose-2,6-bisphosphate. Unphosphorylated fructose-1,6-bisphosphatase was more susceptible to inhibition by both AMP and fructose 2,6-bisphosphate than phosphorylated enzyme, at high and low substrate concentrations. Both ATP and ADP had a similar effect on the two enzyme forms, ADP being the more potent inhibitor. Finally, the combined effect of several inhibitors at physiological concentrations was studied. Under conditions resembling the gluconeogenic state, phosphorylated fructose-1,6-bisphosphatase was found to have twice the activity of the unphosphorylated enzyme.     

A novel mode of Gleevec binding is revealed by the structure of spleen tyrosine kinase

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine --> phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.     

Characterization of the interaction between the N-terminal extension of human cardiac troponin I and troponin C

The N-terminal extension of cardiac troponin I (TnI) is bisphosphorylated by protein kinase A in response to beta-adrenergic stimulation. How this signal is transmitted between TnI and troponin C (TnC), resulting in accelerated Ca(2+) release, remains unclear. We recently proposed that the unphosphorylated extension interacts with the N-terminal domain of TnC stabilizing Ca(2+) binding and that phosphorylation prevents this interaction. We now use (1)H NMR to study the interactions between several N-terminal fragments of TnI, residues 1-18 (I1-18), residues 1-29 (I1-29), and residues 1-64 (I1-64), and TnC. The shorter fragments provide unambiguous information on the N-terminal regions of TnI that interact with TnC: I1-18 does not bind to TnC whereas the C-terminal region of unphosphorylated I1-29 does bind. Bisphosphorylation greatly weakens this interaction. I1-64 contains the phosphorylatable N-terminal extension and a region that anchors I1-64 to the C-terminal domain of TnC. I1-64 binding to TnC influences NMR signals arising from both domains of TnC, providing evidence that the N-terminal extension of TnI interacts with the N-terminal domain of TnC. TnC binding to I1-64 broadens NMR signals from the side chains of residues immediately C-terminal to the phosphorylation sites. Binding of TnC to bisphosphorylated I1-64 does not broaden these NMR signals to the same extent. Circular dichroism spectra of I1-64 indicate that bisphosphorylation does not produce major secondary structure changes in I1-64. We conclude that bisphosphorylation of cardiac TnI elicits its effects by weakening the interaction between the region of TnI immediately C-terminal to the phosphorylation sites and TnC either directly, due to electrostatic repulsion, or via localized conformational changes.     

The effect of fructose 2,6-bisphosphate and AMP on the activity of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase from rat liver

Rat liver fructose-1,6-bisphosphatase was partially phosphorylated in vitro and separated into unphosphorylated and fully phosphorylated enzyme. The effects of fructose 2,6-bisphosphate and AMP on these two enzyme forms were examined. Unphosphorylated fructose-1,6-bisphosphatase was more easily inhibited by both effectors. Fructose 2,6-bisphosphate affected both K0.5 and Vmax, while the main effect of AMP was to lower Vmax. Fructose 2,6-bisphosphate and AMP together acted synergistically to decrease the activity of fructose-1,6-bisphosphatase, and since unphosphorylated and phosphorylated enzyme forms are affected differently, this might be a way to amplify the effect of phosphorylation.     

Tyrosine phosphorylation of the well packed ephrinB cytoplasmic beta-hairpin for reverse signaling. Structural consequences and binding properties

Tyrosine phosphorylation of the 22-residue cytoplasmic region of ephrinB induces its binding to the SH2 domain of Grb4, thus initiating reverse signaling pathways controlling cytoskeleton assembly and remodeling. Recently, the region corresponding to this 22-residue motif was demonstrated to adopt a well packed beta-hairpin structure with a high conformational stability in the unphosphorylated cytoplasmic subdomain. However, because the binding to Grb4 is phosphorylation-dependent and the hairpin contains three conserved tyrosine residues that may be phosphorylated, the key events remain unknown as to how tyrosine phosphorylation affects the structure of this well packed beta-hairpin and which phosphorylation site is relevant to SH2 domain binding. By characterizing the structural and binding properties of six 22-residue SH2 domain-binding motifs with different phosphorylated sites, the present study reveals that, as shown by circular dichroism and NMR, the unphosphorylated 22-residue motif adopts a well formed beta-hairpin structure in isolation from the ephrinB cytoplasmic subdomain. However, this beta-hairpin is radically abolished by tyrosine phosphorylation, regardless of the relative location and number of Tyr residues. Unexpectedly, the peptides with either Tyr304 or Tyr316 phosphorylated show high affinity binding to SH2 domain, whereas the peptide with Tyr311 phosphorylated has no detectable binding. This implies that ephrinB with Tyr311 phosphorylated might have a currently unidentified binding partner distinct from the Grb4 protein, because Tyr311 is known to be phosphorylated in vivo. Based on the results above, it is thus proposed that the disruption of the tight side-chain packing by tyrosine phosphorylation in the well structured region of a signaling protein may represent a general activation mechanism by which a cryptic binding site is disclosed for new protein-protein interactions.     

Molluscan catch muscle myorod and its N-terminal peptide bind to F-actin and myosin in a phosphorylation-dependent manner

Myorod is expressed exclusively in molluscan catch muscle and localizes on the surface of thick filaments together with twitchin and myosin. This protein is an alternatively spliced product of the myosin heavy-chain gene containing the C-terminal rod part of myosin and a unique N-terminal domain. We have recently reported that this unique domain is a target for phosphorylation by gizzard smooth muscle myosin light chain kinase (MLCK) and molluscan twitchin, which contains a MLCK-like domain. To elucidate the role of myorod phosphorylation in catch muscle, a peptide corresponding to the specific N-terminal region of the protein was synthesized in phosphorylated and unphosphorylated form. We report, for the first time, that unphosphorylated full-length myorod and its unphosphorylated N-terminal synthetic peptide are able to interact with rabbit F-actin and thin filaments from molluscan catch muscle. The binding between thin filaments and the peptide was Ca²⁺-dependent. In addition, we found that phosphorylated N-terminal peptide of myorod has higher affinity for myosin compared to the unphosphorylated peptide. Together, these observations suggest the direct involvement of the N-terminal domain of myorod in the regulation of molluscan catch muscle.     

Two apparent molecular weight forms of human and monkey phenylalanine hydroxylase are due to phosphorylation

Two-dimensional polyacrylamide gel analyses of purified human and monkey liver phenylalanine hydroxylase reveal that the enzyme consists of two different apparent molecular weight forms of polypeptide, designated H (Mr = 50,000) and L (Mr = 49,000), each containing three isoelectric forms. The two apparent molecular weight forms, H and L, represent the phosphorylated and dephosphorylated forms of phenylalanine hydroxylase, respectively. After incubation of purified human and monkey liver enzyme with purified cAMP-dependent protein kinase and [gamma-32P]ATP, only the H forms contained 32P. Treatment with alkaline phosphatase converted the phenylalanine hydroxylase H forms to the L forms. The L forms but not the H forms could be phosphorylated on nitrocellulose paper after electrophoretic transfer from two-dimensional gels. Phosphorylation and dephosphorylation of human liver phenylalanine hydroxylase is not accompanied by significant changes in tetrahydrobiopterin-dependent enzyme activity. Peptide mapping and acid hydrolysis confirm that the apparent molecular weight heterogeneity (and charge shift to a more acidic pI) in human and monkey liver enzyme results from phosphorylation of a single serine residue. However, phosphorylation by the catalytic subunit of cAMP-dependent protein kinase does not account for the multiple charge heterogeneity of human and monkey liver phenylalanine hydroxylase.