Dual role for the glutamine phosphoribosylpyrophosphate amidotransferase ammonia channel. Interdomain signaling and intermediate channeling

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalyzes the first reaction of de novo purine nucleotide synthesis in two steps at two sites. Glutamine is hydrolyzed to glutamate plus NH(3) at an N-terminal glutaminase site, and NH(3) is transferred through a 20-A hydrophobic channel to a distal PRPP site for synthesis of phosphoribosylamine. Binding of PRPP is required to activate the glutaminase site (termed interdomain signaling) to prevent the wasteful hydrolysis of glutamine in the absence of phosphoribosylamine synthesis. Mutations were constructed to analyze the function of the NH(3) channel. In the wild type enzyme, NH(3) derived from glutamine hydrolysis was transferred to the PRPP site, and little or none was released. Replacement of Leu-415 at the PRPP end of the channel with an alanine resulted in a leaky channel and release of NH(3) to the solvent. Mutations in five amino acids that line the channel and two other residues required for the reorganization of phosphoribosyltransferase domain "flexible loop" that leads to formation of the channel perturbed channel function as well as interdomain signaling. The data emphasize the role of the NH(3) channel in coupling interdomain signaling and NH(3) transfer.     

Isolation of a multifunctional protein with aminoimidazole ribonucleotide synthetase, glycinamide ribonucleotide synthetase, and glycinamide ribonucleotide transformylase activities: characterization of aminoimidazole ribonucleotide synthetase

5-Aminoimidazole ribonucleotide (AIR) synthetase, glycinamide ribonucleotide (GAR) synthetase, and GAR transformylase activities from chicken liver exist on a single polypeptide of Mr 110,000 [Daubner, C. S., Schrimsher, J. L., Schendel, F. J., Young, M., Henikoff, S., Patterson, D., Stubbe, J., & Benkovic, S. J. (1985) Biochemistry 24, 7059-7062]. Details of copurification of these three activities through four chromatographic steps are reported. The ratios of these activities remain constant throughout the purification. AIR synthetase has an absolute requirement for K+ for activity and under these conditions has apparent molecular weights of 330,000, determined by Sephadex G-200 chromatography, and 133,000, determined by sucrose density gradient ultracentrifugation. Incubation of 18O-labeled formylglycinamidine ribonucleotide (FGAM) with AIR synthetase results in stoichiometric production of AIR, ADP, and [18O]Pi. NMR spectra of beta-FGAM and beta-AIR are reported.     

Glutamine amidotransferase function. Replacement of the active-site cysteine in glutamine phosphoribosylpyrophosphate amidotransferase by site-directed mutagenesis

Site-directed mutagenesis was employed to replace cysteine 12 with phenylalanine in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase). Glutamine-dependent amidophosphoribosyltransferase activity was abolished as a consequence of the mutation. The mutant enzyme, however, exhibited NH3-dependent activity, contained Fe-S, and was normally regulated by AMP. These results document the role of the active site cysteine in activation of glutamine for amide transfer. NH3-dependent amidophosphoribosyltransferase was utilized for de novo purine nucleotide synthesis. Cells containing the mutant enzyme grew at nearly the wild-type rate in media containing a high concentration of NH4Cl. The Phe-12 mutation was used to study NH2-terminal processing. Whereas the wild-type Cys-12 enzyme is processed correctly in Escherichia coli by removal of 11 amino acid residues from the NH2 terminus, the Phe-12 mutant enzyme was not subject to undecapeptide processing. Neither the mutant nor wild-type enzyme made in vitro was correctly processed. Alternative enzymatic and autocatalytic processing mechanisms were considered. The available evidence favors autocatalytic NH2-terminal undecapeptide processing.     

Mutational analysis of the glutamine phosphoribosylpyrophosphate amidotransferase pro-peptide

Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase is synthesized as a pro-enzyme having an 11-amino acid leader. Maturation requires insertion of a [4Fe-4S] cluster and processing of the pro-peptide to expose an NH2-terminal active site cysteine residue. Point and deletion mutations were constructed in the leader region. These mutations affect processing and enzyme activities. Processing of the leader is dependent upon glutamic acid residues at positions -2 and -1 as well as Cys1. In addition, processing requires a pro-peptide longer than 3 residues. Function of the active site cysteine is dependent on pro-peptide processing. Enzyme purified from a pro-peptide deletion strain has activity and iron content that is comparable to the wild type. These results establish that the pro-peptide is not essential for enzyme maturation, but they leave unanswered the question of pro-peptide function.     

A multifunctional protein possessing glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase activities in de novo purine biosynthesis

Three activities on the pathway of purine biosynthesis de novo in chicken liver, namely, glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase, have been found to reside on the same polypeptide chain. Three diverse purification schemes, utilizing three different affinity resins, give rise to the same protein since the final material has identical specific activities for all three enzymatic reactions and a molecular weight on sodium dodecyl sulfate gels of about 110 000. A single antibody preparation precipitates all three activities and binds to the multifunctional protein obtained by two methods in Western blots. Partial chymotryptic digestion of the purified protein gives rise to two fragments, one possessing glycinamide ribonucleotide synthetase activity and the other containing glycinamide ribonucleotide transformylase activity.     

Regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase inactivation in vivo

Glutamine phosphoribosylpyrophosphate amidotransferase is stable in growing cells, but is inactivated in an oxygen-dependent process at various rates in starving or antibiotic-treated cells. On the basis of studies of the purified enzyme, we suggested (D.A. Bernlohr and R.L. Switzer, Biochemistry 20:5675-5681, 1981) that the inactivation in vivo was regulated by substrate stabilization and a competition between stabilizing (AMP) and destabilizing (GMP, GDP, and ADP) nucleotides. This proposal was tested by measuring the intracellular levels of these metabolites under cultural conditions in which the stability of the amidotransferase varied. The results established that the stability of amidotransferase in vivo cannot be explained by the simple interactions observed in vitro. Metabolite levels associated with stability of the enzyme in growing cells did not confer stability under other conditions, such as ammonia starvation or refeeding of glucose-starved cells. The data suggest that a previously unrecognized event, possibly a covalent modification of amidotransferase, is required to mark the enzyme for oxygen-dependent inactivation.     

Role of NifS in maturation of glutamine phosphoribosylpyrophosphate amidotransferase.

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is synthesized as an inactive precursor that requires two maturation steps: incorporation of a [4Fe-4S] center and cleavage of an 11-residue NH2-terminal propeptide. Overproduction from a multicopy plasmid in Escherichia coli leads to the formation of soluble proenzyme and mature enzyme forms as well as a small fraction of insoluble proenzyme. Heterologous expression of Azotobacter vinelandii nifS from a compatible plasmid increased the maturation of the soluble proenzyme three- to fourfold without influencing the content of the insoluble fraction. These results support a role for NifS in heterologous Fe-S cluster assembly and enzyme maturation.     

Oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase in vitro inactivation.

The oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase (ATase) is demonstrated in cell extracts of Bacillus subtilis. The rate of inactivation of ATase in vitro is apparently first order with respect to oxygen concentration and ATase activity. ATase inactivation in vitro (or in vivo) cannot be reactivated by a variety of reductants. ATase is significantly stabilized to oxygen-dependent inactivation in vitro in the presence of tetrasodium phosphoribosylpyrophosphate and glutamine together. The effects of the end product inhibitors, adenosine 5-monophosphate (AMP) and guanosine 5-monophosphate (GMP), on the stability of ATase are antagonistic. AMP stabilizes ATase, whereas GMP destabilizes the enzyme. The stability of ATase can be manipulated over wide ranges by variations in the AMP/GM ratio. The effects of AMP and GMP on the inactivation of ATase in vitro are very specific. ATase is partially inhibited by 1,10-phenanthroline, suggesting that the enzyme contains iron (or some other chelatable metal ion). The inactivation of ATase in vitro is proposed to present a model for the reconstruction of the inactivation of ATase in stationary-phase cells of B. subtilis.     

Oxidation-reduction properties of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

Native Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase contains a [4Fe-4S] cluster in the diamagnetic (+2) state. The cluster is essential for catalytic function, even though amidotransferase does not catalyze a redox reaction. The ability of the Fe-S cluster to undergo oxidation and reduction reactions and the consequences of changes in the redox state of the cluster for enzyme activity were studied. Treatment of the enzyme with oxidants resulted in either no reaction or complete dissolution of the Fe-S cluster and loss of activity. A stable +3 oxidation state was not detected. A small amount of paramagnetic species, probably an oxidized 3Fe cluster, was formed transiently during oxidation. The native cluster was poorly reduced by dithionite, but it could be readily reduced to the +1 state by photoreduction with 5-deazaflavin and oxalate. The reduced enzyme did not display an EPR spectrum typical of [4Fe-4S] ferredoxins in the +1 state, unless it was prepared under denaturing conditions. M?ssbauer spectroscopy of reduced 57Fe-enriched amidotransferase confirmed that the cluster was in the +1 state, but the magnetic properties of the reduced cluster observed at 4.2 K indicated that it is characterized by a ground state spin S greater than or equal to 3/2. The midpoint potential of the +1/+2 couple was too low to measure accurately by conventional techniques, but it was below -600 mV, which is 100 mV more negative than reported for [4Fe-4S] clusters in bacterial ferredoxins. Fully reduced amidotransferase had about 40% of the activity of the native enzyme in glutamine-dependent phosphoribosylamine formation. The fact that both the +1 and +2 forms of the enzyme are active indicates that the cluster does not function as a site of reversible electron transfer during catalysis.     

Amino-terminal deletions define a glutamine amide transfer domain in glutamine phosphoribosylpyrophosphate amidotransferase and other PurF-type amidotransferases.

A series of deletions was constructed in cloned Escherichia coli purF encoding glutamine phosphoribosylpyrophosphate amidotransferase. These deletions extended into the NH2 terminus of the protein and removed amino acids that are required for glutamine-dependent enzyme activity. Enzyme function, ascribed to the NH3-dependent activity, was retained in deletions that removed up to 237 amino acids. This result supports a model in which PurF-type amidotransferases contain an NH2-terminal glutamine amide transfer domain of approximately 194 to 200 amino acids fused to an aminator domain with NH3-dependent function.     

Spectroscopic characterization of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

The properties of the [4Fe-4S] cluster in glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis have been investigated using low temperature magnetic circular dichroism, electron paramagnetic resonance (EPR), and resonance Raman spectroscopies. The Raman spectra of the native enzyme in the Fe-S stretching region show a [4Fe-4S]2+ cluster that is structurally very similar to those in simple redox proteins. Photochemical reduction mediated by 5-deazaflavin with oxalate as the electron donor resulted in [4Fe-4S]+ clusters with a mixture of ground state spin multiplicities. Magnetic circular dichroism and EPR studies of samples ranging in concentration from 0.15 to 0.4 mM concur in finding S = 3/2 [4Fe-4S]+ clusters with predominantly axial and positive zero field splitting as the dominant species. The EPR studies also revealed minor contributions from S = 1/2 [4Fe-4S]+ centers and an S = 5/2 species. The latter becomes the dominant component in more concentrated samples (approximately 2 mM), and arguments are presented in favor of assignment to S = 5/2 [4Fe-4S]+ clusters rather than adventitiously bound high spin Fe(III) ions. The concentration-dependent spin state heterogeneity of the [4Fe-4S]+ cluster in glutamine phosphoribosylpyrophosphate amidotransferase is discussed in light of the magnetic and electronic properties of the [4Fe-4S]+ centers in other enzymes and proteins.     

Involvement of the stringent response in degradation of glutamine phosphoribosylpyrophosphate amidotransferase in Bacillus subtilis

Glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of purine biosynthesis, has previously been shown to be rapidly inactivated and degraded in Bacillus subtilis cells at the end of growth. The loss of enzyme activity appears to involve the oxidation of an iron-sulfur cluster in the enzyme. The degradation of the inactive enzyme involves some elements of the stringent response because it is inhibited in relA and relC mutants. Intracellular pools of guanosine tetra- and pentaphosphate were measured by an improved extraction procedure in cells that had been manipulated in various ways to induce or inhibit amidotransferase degradation. The results are consistent with the hypothesis that one or both of these nucleotides stimulates the synthesis of a protein involved in degradation. An elevated level of these nucleotides was not required for the continued degradation of amidotransferase once it had begun.     

Oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase in stationary-phase cultures of Bacillus subtilis.

Glutamine phosphoribosylpyrophosphate amidotransferase (ATase) activity is rapidly inactivated in stationary-phase cells of Bacillus subtilis. The inactivation of APase requires both the cessation of rapid cell growth and the presence of oxygen. ATase is inactivated in two protease-deficient mutant strains at a rate similar to that seen in the wild type, and is stable in anaerobic cell-free extracts of the parent strain. These results suggest that the inactivation of ATase is not the result of general proteolysis. The inactivation of ATase in stationary-phase cultures can be inhibited by oxygen starvation. This oxygen requirement does not reflect a dependence on the generation of metabolic energy, but appears to be a direct requirement for molecular oxygen. ATase synthesis is repressed by the addition of adenosine, and is inactivated only after the cessation of exponential growth. Addition of chloramphenicol or rifampin to exponential- and stationary-phase cells does not inhibit ATase inactivation, suggesting that protein or ribonucleic acid synthesis is not required for inactivation. ATase is inactivated at the end of exponential growth in cells that have exhausted a required amino acid.     

Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with oxygen: chemistry and regulation by ligands

The inactivation of glutamine phosphoribosylpyrophosphate amidotransferase by reaction of its iron-sulfur center with O2 is believed to be a physiologically important mode of regulation of this enzyme in Bacillus subtilis cells in the stationary phase of growth. Chemical and physical changes accompanying oxidation of the purified enzyme by O2 were studied. The iron of the 4Fe-4S center was oxidized to enzyme-bound high-spin Fe3+; the S2- was oxidized to a mixture of S0 bound as thiocystine and unidentified products. The oxidant appeared to be O2, rather than peroxide, superoxide, hydroxyl radical, or singlet oxygen. Gross physical changes in the oxidized enzyme were shown by its aggregation, decreased solubility, and altered circular dichroic spectrum. Experimental variables affecting the rate of oxidative inactivation were described; the most important of these was modulation of rates of inactivation by the allosteric inhibitors AMP, ADP, GMP, GDP and by the substrate P-Rib-PP. AMP was a potent stabilizer, whose effect was antagonized by P-Rib-PP. The other nucleotides, either acting singly or acting as synergistic pairs, were destabilizers and able to antagonize stabilization by AMP. The results are discussed in terms of the regulation of the stability of amidotransferase and its degradation in vivo.     

A study in molecular contingency: glutamine phosphoribosylpyrophosphate amidotransferase is a promiscuous and evolvable phosphoribosylanthranilate isomerase

The prevalence of paralogous enzymes implies that novel catalytic functions can evolve on preexisting protein scaffolds. The weak secondary activities of proteins, which reflect catalytic promiscuity and substrate ambiguity, are plausible starting points for this evolutionary process. In this study, we observed the emergence of a new enzyme from the ASKA (A Complete Set of E. coli K-12 ORF Archive) collection of Escherichia coli open reading frames. The overexpression of (His)₆-tagged glutamine phosphoribosylpyrophosphate amidotransferase (PurF) unexpectedly rescued a ΔtrpF E. coli strain from starvation on minimal media. The wild-type PurF and TrpF enzymes are unrelated in sequence, tertiary structure and catalytic mechanism. The promiscuous phosphoribosylanthranilate isomerase activity of the ASKA PurF variant apparently stems from a preexisting affinity for phosphoribosylated substrates. The relative fitness of the (His)₆-PurF/ΔtrpF strain was improved 4.8-fold to nearly wild-type levels by random mutagenesis of purF and genetic selection. The evolved and ancestral PurF proteins were purified and reacted with phosphoribosylanthranilate in vitro. The best evolvant (k_cat/K_M = 0.3 s^− 1 M^− 1) was ∼ 25-fold more efficient than its ancestor but > 10⁷-fold less efficient than the wild-type phosphoribosylanthranilate isomerase. These observations demonstrate in quantitative terms that the weak secondary activities of promiscuous enzymes can dramatically improve the fitness of contemporary organisms.     

Chemical genetic identification of glutamine phosphoribosylpyrophosphate amidotransferase as the target for a novel bleaching herbicide in Arabidopsis

A novel phenyltriazole acetic acid compound (DAS734) produced bleaching of new growth on a variety of dicotyledonous weeds and was a potent inhibitor of Arabidopsis (Arabidopsis thaliana) seedling growth. The phytotoxic effects of DAS734 on Arabidopsis were completely alleviated by addition of adenine to the growth media. A screen of ethylmethanesulfonate-mutagenized Arabidopsis seedlings recovered seven lines with resistance levels to DAS734 ranging from 5- to 125-fold. Genetic tests determined that all the resistance mutations were dominant and allelic. One mutation was mapped to an interval on chromosome 4 containing At4g34740, which encodes an isoform of glutamine phosphoribosylamidotransferase (AtGPRAT2), the first enzyme of the purine biosynthetic pathway. Sequencing of At4g34740 from the resistant lines showed that all seven contained mutations producing changes in the encoded polypeptide sequence. Two lines with the highest level of resistance (125-fold) contained the mutation R264K. The wild-type and mutant AtGPRAT2 enzymes were cloned and functionally overexpressed in Escherichia coli. Assays of the recombinant enzyme showed that DAS734 was a potent, slow-binding inhibitor of the wild-type enzyme (I(50) approximately 0.2 microm), whereas the mutant enzyme R264K was not significantly inhibited by 200 microm DAS734. Another GPRAT isoform in Arabidopsis, AtGPRAT3, was also inhibited by DAS734. This combination of chemical, genetic, and biochemical evidence indicates that the phytotoxicity of DAS734 arises from direct inhibition of GPRAT and establishes its utility as a new and specific chemical genetic probe of plant purine biosynthesis. The effects of this novel GPRAT inhibitor are compared to the phenotypes of known AtGPRAT genetic mutants.     

Nucleotide sequence of Escherichia coli purF and deduced amino acid sequence of glutamine phosphoribosylpyrophosphate amidotransferase

The Escherichia coli gene purF, coding for 5-phosphoribosylamine:glutamine pyrophosphate phosphoribosyltransferase (amidophosphoribosyltransferase) was subcloned from a ColE1-purF plasmid into pBR322. Amidophosphoribosyltransferase levels were elevated more than 5-fold in the ColE1-purF plasmid-bearing strain compared to the wild type control, and a further 10- to 13-fold elevation was observed in several pBR322 derivatives. The nucleotide sequence of a 2478-base pair PvuI-HinfI fragment encoding purF was determined. The purF45 structural gene codes for a 56,395 Mr protein chain having 504 amino acid residues. Methionine-1 is removed by processing in vivo leaving cysteine as the NH2-terminal residue. The deduced amino acid sequence was confirmed by comparisons with the NH2-terminal amino acid sequence determined by automated Edman degradation (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536) and amino acid analyses of CNBr peptides including a 4-residue peptide from the CO2H terminus of the enzyme. Nucleotide sequences characteristic of bacterial promoter-operator regions were identified in the 5' flanking region. The coding region appears to be preceded by a 277-297 nucleotide mRNA leader. A deletion removing the putative promoter-operator region results in defective purF expression.     

Measurement of glycinamide ribonucleotide synthetase activity in intact human fibroblasts and Chinese hamster ovary cells

An intact cell assay system based on Tween 80 permeabilization was used to investigate glycinamide ribonucleotide (GAR) synthetase activity in human fibroblasts and Chinese hamster ovary cells. Optimal conditions for the assay of the enzyme were determined with regards to ATP, MgCl2, NH4Cl and ribose-5'-phosphate concentrations as well as pH. Using the optimal assay conditions, the Vmax values as determined by Lineweaver-Burke double reciprocal plots were found to be 5.19 nmol GAR formed/5 X 10(5) cells/30 min for the fibroblasts and 13.4 nmol GAR formed/5 X 10(5) cells/30 min for the Chinese hamster ovary cells.     

Cloning and expression of avian glutamine phosphoribosylpyrophosphate amidotransferase. Conservation of a bacterial propeptide sequence supports a role for posttranslational processing

The cDNA for glutamine phosphoribosylpyrophosphate amidotransferase, the regulatory enzyme of de novo purine nucleotide biosynthesis, has been cloned for the first time from an animal. The derived amino acid sequence of the avian amidotransferase is homologous with amidotransferase sequences from bacteria and yeast. An 11-amino acid propeptide in Bacillus subtilis amidotransferase is conserved in the avian enzyme. Expression in Chinese hamster ovary (CHO) cells and Escherichia coli provides evidence for two post-translational maturation steps needed for synthesis of active enzyme: incorporation of an iron component and processing of the 11-amino acid propeptide. Functional complementation of a CHO amidotransferase mutant suggests that both maturation steps take place in CHO cells. In contrast, function in E. coli requires deletion of the sequence encoding the propeptide. Defective assembly of the iron component may restrict propeptide removal and activation of the avian amidotransferase in E. coli.     

Evidence that the iron-sulfur cluster of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in vivo

Bacillus subtilis glutamine P-Rib-PP amidotransferase contains a [4Fe-4S] cluster which is essential for activity. The enzyme also undergoes removal of 11 NH2-terminal residues from the primary translation product in vivo to form the active enzyme. It has been proposed that oxidative inactivation of the FeS cluster in vivo is the first step in degradation of the enzyme in starving cells. Four mutants of amidotransferases that alter cysteinyl ligands to the FeS cluster or residues adjacent to them have been prepared by site-directed mutagenesis, expressed in Escherichia coli, and characterized (Makaroff, C. A., Paluh, J. L., and Zalkin, H. (1986) J. Biol. Chem. 261, 11416-11423). These mutations were integrated into the B. subtilis chromosome in place of the normal purF gene. Inactivation and degradation in vivo of wild type and mutant amidotransferases were characterized in these integrants. Mutants FeS1 (C448S) and FeS2 (C451S) failed to form active enzyme, assemble FeS clusters, or undergo NH2-terminal processing. The immunochemically cross-reactive protein produced by both mutants was degraded rapidly (t1/2 = 16 min) in exponentially growing cells. In contrast the wild type enzyme was stable in growing cells, and activity and cross-reactive protein were lost from glucose-starved cells with a t1/2 of 57 min. Mutant FeS3 (F394V) contained an FeS cluster and was processed normally, but had only about 40% of normal specific activity. The FeS3 enzyme was also inactivated by reaction with O2 in vitro about twice as fast as the wild type. The amidotransferase produced by the FeS3 integrant was stable in growing cells but was inactivated and degraded in glucose-starved cells more rapidly (t1/2 = 35 min) than the wild type enzyme. Mutant FeS4 (C451S, D442C) also contained an FeS cluster and was processed; the enzyme had about 50% of wild type-specific activity and reacted with O2 in vitro at the same rate as the wild type. Inactivation and degradation of the FeS4 mutant in vivo in glucose-starved cells proceeded at a rate (t1/2 = 45 min) that was somewhat faster than normal. The correlation between absence of an FeS cluster or enhanced lability of the cluster to O2 and increased degradation rates in vivo supports the conclusions that stability of the enzyme in vivo requires an intact FeS cluster and that O2-dependent inactivation is the rate-determining step in degradation of the enzyme. The fact that mutant FeS3 was processed normally but degraded rapidly argues against a role for NH2-terminal processing in controlling degradation rates.