Catalysis of ester hydrolysis by N-(2-dimethylaminoethyl)acetohydroxamic acid (I)

N-(2-dimethylaminoethyl)acetohydroxamic acid was synthesized. This compound, which incorporates a dimethylamino group as a second functionality into the hydroxamic acid molecule, catalyzes the hydrolysis of p-nitrophenyl acetate faster than acetohydroxamic acid itself does. The function of the dimethylamino group is to labilize the intermediate formed in the reaction, thus assisting deacylation intramolecularly. The dimethylamino group carries out this function by intramolecular general base catalysis. Nucleophilic catalysis is ruled out by the sizable deuterium oxide solvent isotope effect () found. General acid-hydroxide ion catalysis is ruled out by determination of the lack of reaction with azide ion, which does not possess a dissociable proton, with the intermediate in this reaction. The deuterium oxide solvent isotope effect on the azide ion reaction of the intermediate also rules out a general acid-hydroxide ion reaction.     

Kinetic alpha-deuterium isotope effects for enzymatic and nonenzymatic hydrolysis of nicotinamide-beta-riboside.

The kinetic α-secondary deuterium isotope effect, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}$, for the pH-independent hydrolysis of nicotinamide riboside, yielding nicotinamide and ribose, in water at 25 ° is 1.14, establishing that this reaction proceeds with unimolecular substrate decomposition to yield a carboxonium ion, or related species, in the rate-determining step. Surprisingly, the corresponding isotope effect for the base-catalyzed decomposition of the same substrate is 1.12, a value indicating considerable sp² character at the Cl′ position in the transition state for this reaction. A similar result, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}\text{= 1.15}$, was obtained for base-catalyzed hydrolysis of NAD⁺. The kinetic alpha deuterium isotope effect for the pig brain NAD glycohydrolasecatalyzed hydrolysis of nicotinamide riboside is 1.08. This value suggests that CN bond cleavage to form an intermediate carboxonium ion, or structurally related species, is at least partially rate-determining. In contrast, the corresponding value for the hydrolysis of this substrate catalyzed by Escherichia coli nicotinamide ribonucleotide glycohydrolase is very near unity, a result consistent with several interpretations including a rate-determining enzyme isomerization reaction.     

On the mechanism of the reaction of tris(hydroxymethyl)aminomethane with activated carbonyl compounds: A model for the serine proteinases

The pH dependence of the reaction of tris(hydroxymethyl)aminomethane (Tris) with the activated carbonyl compound 4-trans-benzylidene-2-phenyloxazolin-5-one (I) is given by the equation $\text{k′}{}_2\text{=}\text{k}{}_{\mathrm{<mtext>b</mtext>}}\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{(K}{}_{\mathrm{<mtext>a</mtext>}}\text{+ [}\text{H}{}^{\mathrm{+}}\text{])}\text{+}\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{[}\text{OH}{}^{\mathrm{?}}\text{]K}{}_{\mathrm{<mtext>a</mtext>}}\text{(K}{}_{\mathrm{<mtext>a</mtext>}}\text{+ [}\text{H}{}^{\mathrm{+}}\text{])}$, where K_a is the dissociation constant of TrisH⁺. Spectrophotometric experiments show that the Tris ester of α-benzamido-trans-cinnamic acid is formed quantitatively over a range of pH values, regardless of the relative contribution of k_b and k_a terms to k′₂. Hence, both terms refer to alcoholysis. While the mechanism of the reaction is not determined unequivocally in the present work, the magnitude of the k_b term, together with its dependence on the basic form of Tris, suggests that ester formation is occurring by nucleophilic attack of a Tris hydroxyl group on the carbonyl carbon of the oxazolinone, with intramolecular catalysis by the Tris amino group. The rate enhancement due to this group is at least 10² and possibly of the order 10⁶. This system is compared with other model systems for the acylation step of catalysis by serine esterases and proteinases.     

The addition of bisulfite to 5-fluorouracil. Evidence for a change in rate determining step

The kinetics of bisulfite addition to 5-fluorouracil were studied as a function of increasing concentrations of potential general acids. Values of $\text{k}{}_{\mathrm{obsd}}\text{[}\text{SO}{}_3{}^{\mathrm{=}}\text{]}$ measured at 25°C and ionic strength 1.0 M increased linearly and then became invariant with increasing concentrations of either HSO₃^? or (OHCH₂CH₂)₂N⁺C(CH₂OH)₃ HCl (BisTris⁺HCl). A small kinetic hydrogen-deuterium isotope effect ($\text{k}{}_{\mathrm{HS}}\text{k}{}_{\mathrm{DS}}\text{= 1.10}$) was observed for the general acid catalysed portion of the addition reaction. The kinetics of bisulfite elimination from 5-fluoro-5,6-dihydrouracil-6-sulfonate were studied in ethanolamine buffers. As previously observed with 1,3-dimethyl-5,6-dihydrouracil-6-sulfonate, this reaction is subject to general base catalysis and exhibits a large kinetic hydrogen-deuterium isotope effect (). The kinetic results for the addition reaction are consistent with a multistep reaction pathway involving the initial formation of an oxyanion sulfite addition intermediate (II) which subsequently adds a proton and undergoes tautomerization to yield the final 5-fluoro-5,6-dihydrouracil-6-sulfonate product. Thus the elimination of bisulfite from 5-fluoro-5,6-dihydrouracil-6-sulfonate probably proceeds by an ElcB mechanism which involves, at relatively low concentrations of general base, rate determining general base catalyzed proton abstraction from carbon 5 to yield intermediate II followed by the rapid elimination of sulfite to yield 5-fluorouracil. These results may be related to both the enzymatically catalyzed dehalogenation of bromoand iodouracil and the methylation of deoxyuridylate by thymidylate synthetase.     

Quantitative structure-activity relationships of chymotrypsin-ligand interactions: An analysis of interactions in p3 space.

Fourteen derivatives of l-alanine of the type CH₃CH(NHCO-3-C₅H₄N)COOR₃ have been synthesized and their hydrolysis by chymotrypsin was studied with the object of characterizing enzymic space (?₃) to which R₃ binds. The binding of R₃ ($\text{log}\text{1}\text{K}{}_{\mathrm{m}}$) was shown via correlation analysis to correlate with molar refractivity (MR) of R₃ rather than hydrophobicity (π). The results confirmed our earlier predictions. A correlation equation for the hydrolysis of 77 acyl-amino acid esters of the general formula R₂CH(NHCOR₁)COOR₃ relating $\text{log}\text{(k}{}_{\mathrm{<mtext>cat</mtext>}}\text{k}{}_{\mathrm{m}}\text{)}$ to molar refractivity of R₁, R₂, and R₃ and to $\text{σ}{}^1$ (Taft's polar parameter) of R₃ was formulated. The general picture of ligand interactions with chymotrypsin as seen with correlation analysis is discussed.     

Model of singlet oxygen scavenging by α-tocopherol in biomembranes

Quenching of singlet molecular oxygen (${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$) by α-tocopherol (I) involves the hydroxy function of the chromanol ring of I. In phosphatidylcholine (PC) uni- and multilamellar vesicles this structural element of I is localized at the interface polar headgroup/hydrophobic core. A dielectric constant of ? ～ 25 was determined for this special region of the PC bilayer. The ratio k_Q/k_R of rate constants of quenching processes (k_Q) and irreversible reactions (k_R) of I with ${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$ increases with decreasing polarity of the solvent. In ethanolic solutions where ? = 25.5, k_Q/k_R is about 40. Extrapolation of these results to phospholipid bilayers suggests that at the nearness of the ester carbonyl oxygen of the PC fatty acid moieties, α-tocopherol can deactivate approximately 40 ${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$ molecules before being destroyed. It is concluded that in vivo, one may expect to find a higher k_Q/k_R ratio if the chromanol ring of I hides within the more hydrophobic interiors of the membrane surface peptides.     

Nuclear magnetic resonance study of the rate of electron transfer between cytochrome c and iron hexacyanides

The rates of electron exchange between ferricytochrome c (C^III)3 and ferrocytochrome c (C^II) were observed as a function of the concentrations of ferrihexacyanide (Fe^III) and ferrohexacyanide (Fe^II) by monitoring the line widths of several proton resonances of the protein. Addition of Fe^II to C^III homogeneously increased the line widths of the two downfield paramagnetically shifted heme methyl proton resonances to a maximal value. This was interpreted as indicating the formation of a stoichiometric complex, C^III·Fe^II, in the over-all reaction:

$\text{C}{}^{\mathrm{III}}\text{+}\text{Fe}{}^{\mathrm{II}}\text{?}\text{k}{}_{\mathrm{?1}}\text{k}{}_1\text{C}{}^{\mathrm{III}}\text{·}\text{Fe}{}^{\mathrm{II}}\text{?}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{C}{}^{\mathrm{II}}\text{·}\text{Fe}{}^{\mathrm{III}}\text{?}\text{k}{}_{\mathrm{?3}}\text{k}{}_3\text{C}{}^{\mathrm{III}}\text{+}\text{Fe}{}^{\mathrm{II}}$

Values for $\text{k}{}_1\text{k}{}_{\mathrm{?1}}\text{= 0.4 × 10}{}^3\text{m}{}^{\mathrm{?1}}\text{and}\text{k}{}_2\text{= 208}\text{s}{}^{\mathrm{?1}}$, respectively, were calculated from the maximal change in line width observed at pH 7.0 and 25 °C. Changes in the line width of C^III in the presence of Fe^II and either KCl or Fe^III suggest that complexation is principally ionic, that Fe^III and Fe^II compete for a common site. Addition of saturating concentrations of Fe^III to C^III produced only minor changes in the nuclear magnetic resonance spectrum of C^III suggesting that complexation occurs on the protein surface.Addition of Fe^III to C^II in the presence of excess Fe^II (to retain most of the protein as C^II) increased the line width of the methyl protons of ligated methionine 80. A value for k_?2 ≈ 2.08 × 10⁴ s^?1 was calculated from the dependence of linewidth on the concentration of Fe^II at 24 °C. These rates are shown to be consistent with the over-all rates of reduction and oxidation previously determined by stopped flow measurements, indicating that k₂ and k_?2 were rate limiting. From the temperature dependence the enthalpies of activation are 7.9 and 15.2 kcal/mol for k₂ and k_?2, respectively.     

Equilibrium constants for association of guanidinium and ammonium ions with oxyanions: The effect of changing basicity of the oxyanion

Using guanidinium and n-butylammonium cations (C⁺) as models for the positively charged side chains in arginine and lysine, we have determined the association constants with various oxyanions by potentiometric titration. For a dibasic acid, H₂A, three association complexes may exist: $\text{K}{}_{\mathrm{<mtext>1M</mtext>}}\text{=}\text{[CHA]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[HA}{}^{\mathrm{?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>1D</mtext>}}\text{=}\text{[CA}{}^{\mathrm{?}}\text{]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[A}{}^{\mathrm{2?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>2D</mtext>}}\text{=}\text{[C}{}_2\text{A]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[CA}{}^{\mathrm{?}}\text{]}$. For guanidinium ion and phosphate, K_1M = 1.4, K_1D = 2.6, and K_2D = 5.1. The data for carboxylates indicate that the basicity of the oxyanion does not affect the association constant: acetate, pK_a = 4.8, K_1M = 0.37; formate, pK_a = 3.8, K_1M = 0.32; and chloroacetate, pK_a = 2.9, K_1M = 0.43, all with guanidinium ion. Association constants are also reported for carbonate, dimethylphosphinate, benzylphosphonate, and adenylate anions.     

Evidence for general catalysis and formation of nitrobenzene in the oxidation of phenylhydroxylamine in aqueous phosphate buffer

N-Phenylhydroxylamine is oxidized in aqueous phosphate buffer to nitrosobenzene, nitrobenzene, and azoxybenzene. Degradation is O₂ dependent and shows general catalysis by $\text{H}{}_2\text{PO}{}_4{}^{\mathrm{?}}$ (k₁ = 2.3 M^?2 sec^?1) and $\text{PO}{}_4{}^{\mathrm{?3}}$ (k₂ = 2.3 × 10⁵M^?2 sec^?1) or kinetically equivalent terms. Evidence is presented suggesting the intermediacy of a highly reactive species leading to these products.     

Isolation and characterization of isocitrate lyase of castor endosperm

Isocitrate lyase (threo-D_S-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: , where A₀ and A_t are activities at time zero at t, and k₁ and k₂ are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pK_a equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg²⁺ (5.0 mm) to a solution of this protein. Addition of Ba²⁺ or Mn²⁺ brings about partial recovery. Other metal ions are not effective.     

The explicit analysis of consecutive pseudo-first-order reactions: Application to kinetics of thiolysis of dithiasuccinoyl (Dts) amino acids

An explicit set of general methods for the experimental determination of the rates k₁ and k₂ of consecutive pseudo-first-order reactions is described and discussed. These rely on the direct simultaneous analytical quantitation of the starting material, intermediate, and product of the reaction, and thus differ from present techniques based on measurement of coreactant consumption or coproduct appearance. The quantity $\text{k}{}^{\mathrm{<mtext>env</mtext>}}\text{=}\text{k}{}_1\text{k}{}_2\text{(k}{}_1\text{+ k}{}_2\text{)}$ is shown to define a good “envelope” approximation to product formation according to the simple law 100% [1 ? exp(?k^envt)]. The theory of envelopes is useful for comparing overall rates of reactions with widely differing values of $\text{κ =}\text{k}{}_2\text{k}{}_1$. The kinetic pattern of thiolysis of dithiasuccinoyl amino acids to carbamoyl disulfide intermediates to product free amino acids is analyzed and shown to agree quantitatively with theory.     

Presteady-state kinetic study of the elementary processes in the chymotrypsin-catalyzed hydrolysis of specific ester substrate. Rate-limiting association process due to the secondary binding.

Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl-l-tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]₀ ? [S]₀ and [S]₀ ? [E]₀ conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant ($\text{k}{}_2\text{K}{}_{\mathrm{s}}\text{′}\text{)}{}_{\mathrm{<mtext>max</mtext>}}\text{= 4.2 × 10}{}^7\text{M}{}^{\mathrm{?1}}\text{s}{}^{\mathrm{?1}}$. The limiting values of K_s′ (dissociation constant of E · S), K₂ (acylation rate) and k₃ (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10^?5 m, 360 ± 15 s^?1 and 29.3 ± 0.8 s^?1, respectively. Likewise small values were observed for K_i of N-(2-furyl)-acryloyl-l-tryptophan and N-(2-furyl)acryloyl-d-tryptophan methyl ester and K_m of N-(2-furyl)acryloyl-l-tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a $\text{k}{}_{\mathrm{?1}}\text{k}{}_2$ value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k₂ value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters.     

Kinetics of bidirectional active sodium fluxes in the toad bladder

The kinetics of isotopic Na⁺ flows was studied in urinary bladders of toads from the Dominican Republic. Initial studies of the potential dependence of passive serosal to mucosal ²²Na⁺ efflux demonstrated the absence of isotope interaction and/or other coupling with passive Na⁺ flow. The electrical current $\text{I}$ and mucosal to serosal ²²Na⁺ influx were then measured with transmembrane potential clamped at $\text{Δψ = 0, 25, 50, 75 or 100}\text{mV}$. Subsequent elimination of active Na⁺ transport mucosal amiloride permitted calculation of the rates of active Na⁺ transport $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}$ and active and passive influx and $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}$ and $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>p</mtext>}}$. The results indicate that for Dominican toad bladders mounted in chambers only Na⁺ contributes significantly to transepithelial active ion transport; hence $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}\text{= J}{}^{\mathrm{<mtext>a</mtext>}}$. $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ was abolished at $\text{Δψ = E = 96.3 ± 1.9}\text{(S.E.) mV}$. As Δψ approached $\text{E}$, active efflux $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ became demonstrable. At $\text{Δ = 100}\text{mV}\text{,}\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ exceeded $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$, so that $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ was negative. Experimental values of $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ agreed well with theoretical values predicted by a thermodynamic formulation: $\text{J}{}_{\mathrm{<mtext>exp</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}\text{= 0.985}\text{J}{}_{\mathrm{<mtext>theor</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}\text{(r = 0.993)}$. The dependence of $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ on Δψ is curvilinear.     

Anomerization of glucose 6-phosphate. pH dependence and solvent isotope effects.

The anomerization of α-d-glucose 6-phosphate has been examined using a spectrophotometric coupled enzyme assay. The pH-rate profile for spontaneous d-glucose 6-phosphate anomerization reveals that the d-glucose 6-phosphate dianion is the species giving rise to the much higher rate of d-glucose 6-phosphate anomerization over that of d-glucose. A deuterium solvent isotope effect of is consistent with the postulated intramolecular general-base catalysis by the phosphate.     

H/2H isotope effect in redox reactions of cytochrome c

The rate of reaction of ferro- and ferricytochrome c (C(II) and C(III)) with ferri- and ferrocyanide and of C(III) with O₂^? and CO₂^? was determined in H₂O and in ²H₂O in the temperature range 5–35 °C. No isotope effect was evident in any of the reductions of C(III); the apparent energy of activation was identical in H₂O and ²H₂O. An isotope effect with , depending on pH for instance was observed in the oxidation of C(II), in the slow phase of oxidation which involves conformational changes. An interpretation (supported by evidence from previous work) involving water molecules in the close vicinity of the reaction site on the protein is discussed.     

Some observations on a new type of point average molecular weight

A new type of (reduced) point average molecular weight A¹, is described. Several interesting properties are developed: (i) $\text{A}{}^1\text{=}\text{reduced}$ weight average molecular weight over the whole cell, $\text{A}{}_{\mathrm{<mtext>w</mtext>}}{}^{\mathrm{o}}\text{A}{}^1\text{(}\text{meniscus}\text{) = A}{}_{\mathrm{<mtext>w</mtext>}}\text{(}\text{meniscus}\text{); (iii) A}{}^1\text{(}\text{zero concentration}\text{) =}\text{reduced}$ number average molecular weight, A_n (meniscus). In addition, its usefulness in extracting the meniscus concentration, J(a), and in examining heterogeneous systems such as mucus glycoproteins, are discussed. The evaluation and application of $\text{A}{}^1$ requires only simple computational facilities, without the use for large-scale multiple data acquisition and recycling techniques.     

Study on models of single populations: an expansion of the logistic and exponential equations

Using the adsorption theory of chemical kinetics, a new equation concerning the growth of single populations is presented:

$\text{d}\text{X}\text{d}\text{t}\text{=}\text{μ}{}_{\mathrm{c}}\text{X(1 ?)}\text{X}\text{X}{}_{\mathrm{m}}\text{1?}\text{X}\text{X}{}_{\mathrm{m}}{}^{\mathrm{\prime }}$

or in its integral form:

$\text{ln}\text{X}\text{X}{}_{\mathrm{o}}\text{?}\text{ln}\text{X}{}_{\mathrm{m}}\text{?X}\text{X}{}_{\mathrm{m}}\text{?X}{}_{\mathrm{o}}\text{+}\text{X}{}_{\mathrm{m}}\text{X}{}^{\mathrm{\prime }}{}_{\mathrm{m}}\text{X}{}_{\mathrm{m}}\text{?X}\text{X}{}_{\mathrm{m}}\text{?X}{}_{\mathrm{o}}\text{=μ}{}_{\mathrm{c}}\text{(t?t}{}_{\mathrm{o}}\text{)}$

This equation attempts to explain the relationship between population increment and limiting resources. It can be reduced to either the logistic or exponential equation under two extreme conditions. The new equation has three parameters, X_m, X′_m and μ_c, each of which has ecological significance. $\text{X}{}_{\mathrm{m}}\text{X′}{}_{\mathrm{m}}$ concerns the efficiency of nutrient utilization by an organism. Its value is between zero and one. With ratios approaching unity, the efficiency is high; lower ratios indicate that population increment is quickly restricted by limiting resources. μ_c, is a velocity parameter lying between μ_e, (exponential growth) and μ_L (logistic growth), and is dependent on the value of $\text{solX}{}_{\mathrm{m}}\text{X′}{}_{\mathrm{m}}$. From μ_c we can predict the time course of population incremental velocity ($\text{d}\text{X}\text{d}\text{t}$), and can observe that it is not symmetrical, unlike that derived from the logistic equation. At $\text{X}{}_{\mathrm{m}}\text{X′}{}_{\mathrm{m}}\text{= 1}$ the maximum velocity of the population increment predicted from the new equation is twice that of the logistic equation.Population growth in nature seems to support the new equation rather than the logistic equation, and it can be successfully fitted by means of a least square method.     

Effects of 5-hydroxylysine on acetylene reduction and NH4+-assimilation in the cyanobacterium Anabaena cylindrica.

5-hydroxylysine, an analogue of glutamate and lysine, causes $\text{NH}{}_4{}^{\mathrm{+}}$ production by N₂-fixing A. cylindrica; it also reversibly inhibits GS activity in vitro but has no effect on alanine dehydrogenase or GOGAT. On adding 5-hydroxylysine intracellular pools of glutamine, glutamate and aspartate decrease; those of alanine and serine increase. 5-hydroxylysine alleviates the inhibitory effect of $\text{NH}{}_4{}^{\mathrm{+}}$ on heterocyst production and C₂H₂ reduction and in $\text{NH}{}_4{}^{\mathrm{+}}\text{-grown}$ cultures results in heterocyst synthesis and in C₂H₂ reduction. The data suggest that the GS-GOGAT pathway is the sole route of importance in primary $\text{NH}{}_4{}^{\mathrm{+}}$ assimilation in A. cylindrica, that $\text{NH}{}_4{}^{\mathrm{+}}$ alone does not inhibit nitrogenase and heterocyst production, and that GS and/or a product is involved in regulating the production of both.